Nicotinic receptor-associated modulation of stimulatory and inhibitory neurotransmitters in NNK-induced adenocarcinoma of the lungs and pancreas

Abstract

Small airway-derived pulmonary adenocarcinoma (PAC) and pancreatic ductal adenocarcinoma (PDAC) are among the most common human cancers and smoking is a risk factor for both. Emerging research has identified cAMP signalling stimulated by the stress neurotransmitters adrenaline and noradrenaline as an important stimulator of adenocarcinomas, including PAC and PDAC. The nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent mutagen and the most powerful tobacco carcinogen. NNK is also an agonist for nicotinic acetylcholine receptors (nAChRs). Using hamster models of NNK-induced PAC and PDAC, we have tested the hypothesis that in analogy to chronic effects of nicotine in the brain, NNK may modulate the alpha(7)- and alpha(4)beta(2)nAChRs, causing an increase in stress neurotransmitters and a decrease in the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Immunoassays showed a significant increase in serum adrenaline/noradrenaline and increased intracellular cAMP in the cellular fraction of blood of NNK-treated hamsters. Western blots on microdissected control small airway epithelia, alveolar epithelia, pancreatic islet and pancreatic duct epithelia, and from NNK-induced PACs and PDACs showed that the GABA-synthesizing enzyme glutamate decarboxylase 65 (GAD65) and GABA were suppressed in NNK-induced PACs and PDACs. In contrast, protein expression of the alpha(7)nAChR, alpha(4)nAChR as well as p-CREB and p-ERK1/2 were up-regulated. These findings suggest that NNK-induced alterations in regulatory nAChRs may contribute to the development of smoking-associated PAC and PDAC by disturbing the balance between cancer-stimulating and -inhibiting neurotransmitters.

Figure 1

Simplified cartoon, summarizing the effects of chronic exposure to nicotine on excitatory and inhibitory neurotransmitter signaling regulated by nicotinic acetylcholine receptors in the nervous system. Chronic exposure to nicotine upregulates the α₇nAChR without desensitizing the receptor[13, 34], resulting in increased release and synthesis of excitatory neurotransmitters glutamate, dopamine, serotonin and noradrenaline stimulated by this receptor. Most of the stimulating neurological and psychological effects of these neurotransmitters are mediated by the activation of adenylyl cyclase downstream of Gα_s-coupled receptors. GABA normally balances these effects by inhibiting adenylyl cyclase downstream of the Gα_i-coupled GABA_BR[13, 54]. The α₄β₂nAChR that stimulates the release and synthesis of GABA is desensitized by chronic exposure to nicotine, thus virtually shutting down all inhibitory GABA signaling[54]. This imbalance in stimulatory and inhibitory brain functions has been implicated in nicotine addiction and withdrawal symptoms [13, 34, 53].

Figure 2

Results of immunoassays demonstrating the modulation of serum adrenaline/noradrenaline levels and cAMP in blood cells of hamsters treated for 20 weeks with NNK. There was a 3.8-fold increase in noradrenaline and a 2.3-fold increase in adrenaline while cAMP was increased 2.2-fold. Data are mean values and standard errors of triplicate samples from the pooled blood of 10 hamsters per treatment group. Asterixes indicate values significantly (p<0.001) different from controls.

Figure 3

Western blots, illustrating the upregulation of α₇- and α₄nAChR protein in NNK-induced PAC. In comparison with their normal cells of origin, small airway epithelial cells (SAEC), α₄subunit protein was increased 3.2-fold (*: p<0.001) and α₇ subunit protein 3.4-fold (*: p<0.001). Data in the graph are mean values and standard errors of ratios of nAChR-protein over actin from five densitometric readings per band and normalized with expression levels in SAECs set at 100%. Each Western blot was conducted three times with similar data.

Figure 4

Western blots, exemplifying the suppression of GAD and GABA in NNK-induced PAC. GAD65 was reduced to 25% (*: p<0.001) and GABA to 26% (*: p<0.001) of the levels observed in SAECs. Data in the graph are normalized (expression levels in SAEC=100%) mean values and standard errors of ratios of GAD or GABA over actin from five densitometric readings per band. Each Western blot was conducted three times with similar data.

Figure 5

Western blots, documenting the induction of p-CREB and p-ERK1/2 in NNK-induced PAC. The increase in p-CREB expression was 1.7-fold (*: p<0.001) and that of p-ERK1/2 1.8-fold (*: p<0.001) over the levels in SAECs. Data in the graph are normalized (expression levels in SAEC=100%) mean values and standard errors of ratios of p-CREB over CREB and p-ERK1/2 over ERK1/2 from five densitometric readings per band. Each Western blot was conducted three times with similar data.

Figure 6

Western blots, showing the upregulation of α₄-and α₇ nAChR subunit proteins in NNK-induced PDAC. Expression levels of the α₄nAChR was increased 2.3-fold (*: p<0.001) and that of the α₇nAChR 1.8-fold (*: p<0.001) over levels observed in duct epithelial cells. Data in the graph are mean values and standard errors of five densitometric readings per band of ratios of nAChR protein over actin and normalized with levels in duct epithelial cells set as 100%. Each Western blot was conducted three times with similar data.

Figure 7

Western blots, exemplifying the suppression of GAD and GABA in NNK-induced PDAC. Both, GAD65 and GABA were reduced to barely detectable levels of below 10% of control duct epithelial cells (*: p<0.001). Quantitative data in the graph are mean values and standard errors of ratios of GAD or GABA over actin and are normalized with levels in duct epithelial cells set at 100%. Each Western blot was conducted three times with similar results.

Figure 8

Western blots, showing the induction of p-CREB and p-ERK1/2 in NNK-induced PDAC. Both phosphorylated proteins were barely detectable in control islet and duct cells. P-CREB was increased 2.65-fold (*: p<0.001) and p-ERK1/2 2.55-fold (*: p<0.001) over the levels observed in duct epithelial cells. Data in the graph are normalized (expression levels in duct cells=100%) mean values and standard errors of ratios of p-CREB over CREB and p-ERK1/2 over ERK1/2 from five densitometric readings per band. Each Western blot was conducted three times with similar data.

Figure 9

Simplified cartoon illustrating the proposed effects of chronic exposure to NNK on neurotransmitter signaling in PAC and PDAC and their microenvironment. Based on the finding that NNK is an nAChR agonist [9], in conjunction with the data reported in this publication, we propose that chronic exposure to this carcinogenic nitrosamine has effects on nAChRs in PAC and PDAC similar to those reported for nicotine in the nervous system (Figure 1). In peripheral non neurogenic tissues [27] and sympathetic nerve endings [55] the α₇nAChR stimulates the release of noradrenaline from which adrenaline is formed. The observed increase in these stress neurotransmitters due to upregulated α₇nAChRs enhances cancer stimulating cAMP signaling [21, 23, 24, 44] downstream of β-adrenergic receptors (β-ARs), an effect further intensified by direct stimulation of these receptors by NNK [21, 23] and by NNK-induced activating point mutations in K-ras [39, 56]. At the same time, inhibition of tumor stimulating cAMP signaling by GABA [37, 47] is virtually shut down due to desensitization of α₄β₂nAChRs.