br Corresponding authors at Department of
Corresponding authors at: Department of Pharmacology and Therapeutics, School of Medicine, Universidad Autónoma de Madrid, Arzobispo Morcillo 4, 28029, Madrid, Spain.
Corresponding authors at: Internal Medicine Service, University Hospital La Paz of Madrid, Paseo de la Castellana 261, 28046, Madrid, Spain. E-mail addresses: [email protected] (F. Arnalich), [email protected] (C. Montiel). 1 These authors contributed equally to this paper as first author.
carcinogens, nicotine, the addictive component of tobacco, and its carcinogenic derivative nitrosamine 4-(methylnitrosamino)-1-(3-pyr-ydyl)-1-butanone (NNK) also contribute to the initiation and progres-sion of lung cancer by activating nicotinic KN 93 receptors (nAChRs) expressed in these tumor cells [4–7].
nAChRs are complex structures having five subunits. Several alpha (α3-α7, α9) and beta (β2 and β4) nAChR subunits have been identified in human NSCLC cell lines and in primary lung tumors from NSCLC patients [see Ref.  and references therein ,]. Therefore, the above tumors can express several nAChR subtypes composed of identical subunits (homomeric α7- and α9-nAChRs) or of diﬀerent α and β subunits (heteromeric α3β4α5-, α3β2α5- and α4β2-nAChRs). Despite this diversity of nAChR subtypes, the α7 subtype is recognized as being the main trigger for the nicotine-mediated proliferative, pro-angiogenic and pro-metastatic eﬀects in human NSCLC [10–13]. These nicotine eﬀects result from α7-nAChR-mediated downstream activation of sev-eral oncogenic signaling pathways that, in the case of NSCLC cells, involve the MEK/ERK, Akt and Rb-Raf-1/phospho-ERK/phospho-p90RSK pathways [see Ref [6,14]. and references therein]. Accord-ingly, the α7-nAChR subtype could be considered as a target for lung cancer prevention and/or therapy.
The α7 subunits that make up the α7-nAChR in humans are encoded by the CHRNA7 gene located on the long arm of chromosome 15 (15q13-q14). This gene is partially duplicated in the same chromosome and, after its fusion with the FAM7A gene, generates a new chimeric gene (CHRFAM7A), which is unique to the human genome since it is not found in other higher primates [15,16]. This chimeric gene encodes for a truncated α7 nAChR subunit form, dupα7, which shares all the structural elements of the ancestral protein except for a substantial part of the N-terminal region containing the signal peptide and the agonist-binding domain . Furthermore, dupα7 and α7 subunits are natu-rally expressed in the same human cell types and tissues, including neurons, epithelial and immune cells [see Ref . and references therein] and primary NSCLC tumors .
The functional role of the above human-specific duplicated gene was long unidentified until we demonstrated that its product negatively regulated α7-nAChR activity in Xenopus oocytes , a finding corro-borated shortly afterwards by others . It is noteworthy that the blocking eﬀect of dupα7 on α7-nAChR function in amphibian cells has also been found in mammalian cells, as we have recently reported in murine macrophages, where dupα7 overexpression attenuated the known anti-inflammatory eﬀect controlled by the α7-nAChRs naturally expressed in these cells . Given the prominent role of α7-nAChRs in tobacco-induced lung cancer progression and also the fact that dupα7 is natively expressed in primary tumors from NSCLC patients , it is feasible that this latter subunit may act as an endogenous suppressor of the growth and progression of the above tumors by interfering with their α7-nAChR responsiveness. To date, there have been no data in the literature on this issue.
We addressed this lack of information by performing in vitro and in vivo tumorigenic assays after stable overexpression of an epitope-tagged dupα7 construct (dupα7.pcDNA3.1/Myc-His) in human NSCLC cell lines (A549 and SK-MES-1) and in A549 xenografts in nude mice. The construct, prepared in our laboratory, is crucial for the selection of cells with stable dupα7-Myc transfection since, otherwise, the high homology in the peptide sequences between the dupα7 and α7 subunits makes commercially-available antibodies cross-react with both sub-units, preventing the immunocytochemical identification and selection of clones with stable dupα7 overexpression.
Here, we demonstrate the anti-tumor activity of the duplicated form of the α7 subunit by blocking, both in vitro and in vivo, the tobacco-promoted oncogenic eﬀect of the ancestral protein that makes up the α7-nAChR. Lung Cancer 128 (2019) 134–144
2. Material and methods
2.1. Cell cultures and reagents
Human NSCLC cell lines (A549 and SK-MES-1) were purchased from American Type Culture Collection (ATCC) and maintained respectively in RPMI 1640 (Gibco, Thermo Fischer Scientific, Waltham, MA, USA) or MEM (Sigma-Aldrich; St. Louis, USA) media supplemented with 10% fetal bovine serum (FBS) in a 5% CO2 humidified incubator at 37 °C. Nicotine and NNK were purchased from Sigma-Aldrich and Toronto Research Chemicals (ON, Canada), respectively. The following primary antibodies were used: mouse anti-Myc (Roche, Mannheim, Germany); rabbit anti-β-catenin (Cell Signaling Technology, Danvers, MA, USA); mouse anti-vimentin, mouse anti-fibronectin, goat anti-β-actin, rabbit anti-VEGF and rabbit anti-α7 (Santa Cruz Biotechnologies, CA, USA); and mouse anti-Ki67 (Dako, Santa Clara, CA, USA).