Cholinergic Targets in Lung Cancer

Abstract

Lung cancers express an autocrine cholinergic loop in which secreted acetylcholine can stimulate tumor growth through both nicotinic and muscarinic receptors. Because activation of mAChR and nAChR stimulates growth; tumor growth can be stimulated by both locally synthesized acetylcholine as well as acetylcholine from distal sources and from nicotine in the high percentage of lung cancer patients who are smokers. The stimulation of lung cancer growth by cholinergic agonists offers many potential new targets for lung cancer therapy. Cholinergic signaling can be targeted at the level of choline transport; acetylcholine synthesis, secretion and degradation; and nicotinic and muscarinic receptors. In addition, the newly describe family of ly-6 allosteric modulators of nicotinic signaling such as lynx1 and lynx2 offers yet another new approach to novel lung cancer therapeutics. Each of these targets has their potential advantages and disadvantages for the development of new lung cancer therapies which are discussed in this review.

Figure 1

Cholinergic signaling pathways in lung cancers. Diagram of pathways for cholinergic signaling and potential points where signaling can be interrupted. Choline is taken into cells; acetylcholine (ACh) synthesized by the action of choline acetyltransferase (ChAT), packaged and secreted to interact with nAChR and mAChR receptors on the same or neighboring cells. Muscarinic and nicotinic receptors can also be activated by ACh from neighboring or distal sources. Nicotinic receptors can be activated by nicotine and up- or down-regulated by both membrane bound ly-6 proteins such as lynx1 or lynx2 or secreted ly-6 proteins such as slurp-1.

Figure 2

Cholinergic modification of lung cancer cell growth. H82 small cell lung carcinoma cells were plated in 96-well culture plates and cell proliferation measured after specified drug treatments. Cell numbers were measured at 0, 6, 12 days using the MTS assay. A. The nicotinic agonist nicotine; B. The muscarinic agonist carbachol; C. The nicotinic antagonist mecamylamine; D. The muscarinic antagonist atropine; E. The choline transport inhibitor hemicholinium-3; F. The vesicular acetylcholine transporter inhibitor (VAChT) vesamicol. * p<0.05 by Neuman-Keuls test following ANOVA. All data are expressed as the mean ± SE of twelve replicates. Drug concentrations as shown in panel A. Modified after Song et al [12].

Figure 3

α7 nAChR and lung cancer cell growth. A. The α7 nAChR antagonist methyllycaconitine (MLA) has no effect on cell growth of A549 lung adenocarcinoma cells. Similar results were seen with α-bungarotoxin and in the H82 and H520 cell lines (data not shown). B. siRNA knockdown of α7 decreases cell growth in H520 squamous cell lung carcinoma cells compared to cells transfected with control siRNA. C. siRNA knockdown of α7 decreases cell growth in A549 lung adenocarcinoma cells compared to cells transfected with control siRNA.

Figure 4

Muscarinic modulation of lung cancer cell growth. A. The M3 antagonist darifenacin blocks ACh-induced increase in intracellular Ca⁺⁺ in H82 SCLC cells. B. The M3 mAChR antagonist 4-DAMP inhibited H82 cell proliferation in a concentration-dependent manner. * p < 0.001 and † p < 0.05 compared to control at 9 days by Tukey-Kramer multiple comparison test after Two-way ANOVA. C. Effect of the M3-antagonist darifenacin on growth of H82 tumor xenografts in nude mice. Tumor volume. * p < 0.05 compared to control at same time point by Tukey-Kramer multiple comparison test after repeated measures ANOVA. D. Effect of darifenacin on MAPK and Akt phosphorylation in the tumor xenografts. Ratio of density of phosphorylated to unphosphorylated Akt and MAPK in H82 tumor xenografts is shown along with representative bands from western blots for each treatment. Modified after Song et al [58].

Figure 5. Alignment of Lynx1, lynx2 and PSCA ly-6 proteins

Proteins were aligned using the Clustal Omega alignment tool. The conserved cysteine residues that define the family are shown by a triangle. The putative GPI cleavage site as predicted by PredGPI is boxed [78]. * indicates fully conserved amino acids other than Cysteine, : indicates conservation of strongly similar amino acids, . indicates conservation of weakly similar amino acids. The secreted ly-6 proteins slurp-1 and slurp-2 are not included in the alignment, but if they were they would lack the C-terminal hydrophobic residues and the GPI cleavage site, but would still maintain the conserved cysteine residues.

Figure 6. Lynx1 expression and function in lung cancers cells

A. Relative RNA levels of Lynx1 in squamous cell carcinomas compared to normal and also plotted according to degree of differentiation. †p < 0.03 by t-test, *p < 0.05 compared to normal tissue by Tukey-Kramer after One-way ANOVA. Number of samples of each grade is shown in figure legend in parentheses. Figure modified after Song et al [13]. B. siRNA knockdown of lynx1 increased cell growth of A549 lung adenocarcinoma cells. Levels of lynx1 were decreased by ~70% by the siRNA knockdown (data not shown). Data are mean ± SD of 20 replicates in two separate experiments. * p < 0.05 versus control siRNA. C. Effect of increased lynx1 expression in A549 cells. A lentivirus expressing lynx1 was prepared and A549 cells transduced with the lynx-1 lentiviral vector or a control lentivirus. Lynx1 protein expression was highly expressed after transduction (data not shown). Cell growth is shown relative to day 0. * p < 0.05 compared to controls. From Fu et al [76].