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journal homepage: www.elsevier.com/locate/lungcan
Anti-tumoral activity of the human-specific duplicated form of α7-nicotinic T receptor subunit in tobacco-induced lung cancer progression
José Luis Cedilloa,1, Anna Bordasa,1, Francisco Arnalichb, , Isabel Esteban-Rodríguezc, Carolina Martín-Sáncheza, María Extremeraa, Gema Atienzaa, Juan J. Riosb, Raquel L. Arribasa, Carmen Montiela,
a Department of Pharmacology and Therapeutics, School of Medicine, Universidad Autónoma de Madrid-IdiPAZ, Madrid, Spain
b Internal Medicine Service, University Hospital La Paz of Madrid-IdiPAZ, Madrid, Spain
c Pathology Service, University Hospital La Paz of Madrid-IdiPAZ, Madrid, Spain
Keywords: α7 Nicotinic VX-765 receptor dupα7 Nicotinic receptor subunit Non-small cell lung cancer
Objectives: Tobacco smoking is strongly correlated with the onset and progression of non-small cell lung cancer (NSCLC). By activating α7 nicotinic acetylcholine receptors (α7-nAChRs) in these tumors nicotine and its to-bacco-derived nitrosamine, NNK, contribute to these oncogenic processes. Here, we investigated whether the human-specific duplicated form of the α7-nAChR subunit (dupα7) behaves as an endogenous negative regulator of α7-nAChR-mediated tumorigenic activity induced by tobacco in NSCLC cells, similarly to its influence on other α7-nAChR-controlled functions in non-tumor cells.
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 acetylcholine 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