br Methods Two human NSCLC cell
Methods: Two human NSCLC cell lines, lung adenocarcinoma (A549) and squamous cell carcinoma of the lung (SK-MES-1), both wild-type or with stable overexpression of dupα7 (A549dupα7 or SK-MES-1dupα7), were used to investigate in vitro anti-tumor activity of dupα7 on nicotine- or NNK-induced tumor progression. For this purpose, migration, proliferation or epithelial-mesenchymal transition (EMT) were examined. The anti-tumor eﬀect of dupα7 on nicotine-promoted tumor growth, proliferation or angiogenesis was also assessed in vivo in an athymic mouse model implanted with A549dupα7 or A549 xenografts. Results: Overexpression of dupα7 in both cell lines almost completely suppresses the in vitro tumor-promoting eﬀects induced by nicotine (1 μM) or NNK (100 nM) in wild-type cells. Furthermore, in mice receiving nicotine, A549dupα7 xenografts show: (i) a significant reduction of tumor growth, and (ii) decreased expression of cell markers for proliferation (Ki67) or angiogenesis (VEGF) compared to A549 xenografts.
Conclusion: Our study demonstrates, for the first time, the in vitro and in vivo anti-tumor capacity of dupα7 to block the α7-nAChR-mediated tumorigenic eﬀects of tobacco in NSCLC, suggesting that up-regulation of dupα7 expression in these tumors could oﬀer a potential new therapeutic target in smoking-related cancers.
Lung cancer is the leading global cause of cancer deaths, with non-small cell lung cancer (NSCLC) accounting for 75–85% of all lung cancer cases . Lung adenocarcinoma and squamous cell carcinoma of
the lung are the two major histological types of NSCLC. Cigarette smoking is an important risk factor for many types of cancers, including NSCLC, which is understandable because tobacco smoke contains more than 70 known carcinogens that will eventually initiate carcinogenesis [2,3]. In parallel with the mutagenic and cytotoxic eﬀects of these
Abbreviations: α7-nAChR, α7 nicotinic (±)-Baclofen receptor subtype; dupα7, human-specific duplicated form of the α7-nAChR subunit; EMT, epithelial-me-senchymal transition; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; HRP, horseradish peroxidase; IHC, immunohistochemistry; MEK, mitogen-activated protein kinase; NNK, nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyrydyl)-1-butanone; NSCLC, non-small cell lung cancer; p90RSK, MAPK-activated protein kinase-1; PVDF, polyvinylidene difluoride; qPCR, real-time quantitative PCR; Raf-1, RAF proto-oncogene serine/threonine-protein kinase; Rb, retinoblastoma tumor suppressor protein; SCLC, small cell lung cancer; VEGF, vascular endothelial grown factor
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 acetylcholine 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.