Abstract LB-110: Mechanisms underlying α5 nicotinic receptor polymorphisms and increased lung cancer risk: implications for therapy with nicotinic antagonists
Authors: Spindel Eliot R. , Rekow Stephen S. 
Published: May 2015

GWAS studies have linked the 18q25 nicotinic receptor locus that contains the α3, α5 and β4 nicotinic acetylcholine receptors (nAChR) to increased risk of lung cancer. In that locus, three independent groups of polymorphisms are linked to increased risk of lung cancer. Two of the groups are associated with increased levels of α5 and one group is linked to rs16969968 which is a single nucleotide polymorphism in which amino acid 398 of α5 is changed from Asp (D) to Asn (N). Our laboratory has previously shown that almost all lung cancers express nAChR and nicotine can stimulate lung cancer cell growth through nAChR-dependent mechanisms. Now using combinations of shRNA knockdown of α5 and lentiviral transduction of either α5-398D or α5-398N we have explored the mechanisms by which α5 expression and structure might increase lung cancer risk. First α5 was knocked down in the A549 lung adenocarcinoma cell line using the lentiviral vector pPrime expressing shRNAs directed against two different sequences of α5. Efficiency of knockdown was greater than 90%. Control cells were transduced with a pPrime vector expressing luciferase and these cells showed no change in levels of alpha5. Effects on basal and nicotine-stimulated cell growth were measured using a soft agar clonogenic assay. In control A549 cells, both 3 and 10 μM nicotine significantly increased colony formation. In α5 knockdown cells there was a small decrease in basal cell growth, but nicotine lost all ability to stimulate cell growth and even caused a small decrease in cell growth. Thus in the absence of α5 nAChR, nicotine no longer stimulates lung cancer cell growth. Next the A549 cells in which α5 had been knocked down were transduced with either α5-398D or α5-398N (constructs were designed not to be targeted by the α5 shRNA used to knockdown endogenous α5). In cells transduced with α5-398D, nicotine stimulated cell colony formation similarly to control cells. By contrast in cells transduced with α5-398N, nicotine failed to stimulate colony formation. This is consistent with studies in neuronal systems that show the α5-398N mutant shows smaller responses to nicotine than does α5-398D. The failure of the α5-398N to stimulate colony formation in response to nicotine strongly suggests that the linkage of the α5-398N SNP to increased lung cancer risk stems from the increased smoking associated with the SNP rather than a direct effect on lung cancer growth. Taken together this data suggests that α5 antagonists will block the ability of nicotine to stimulate lung cancer growth in patients with SNPs associated with increased levels of α5 but have little effect on the association of the α5-398N SNP with lung cancer. In addition, in that α5 knockdown has been shown to increase nicotine intake, if α5 antagonists were used for lung cancer therapy or chemoprevention, inhaled antagonists that did not cross the blood brain barrier would be optimal.

Citation Format: Eliot R. Spindel, Stephen S. Rekow. Mechanisms underlying α5 nicotinic receptor polymorphisms and increased lung cancer risk: implications for therapy with nicotinic antagonists. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-110. doi:10.1158/1538-7445.AM2014-LB-110