Forum Minireview. Pingfang Song 1 and Eliot R. Spindel 1, *

Transcription

1 J Pharmacol Sci 106, (2008)2 Journal of Pharmacological Sciences 2008 The Japanese Pharmacological Society Forum Minireview Basic and Clinical Aspects of Non-neuronal Acetylcholine: Expression of Non-neuronal Acetylcholine in Lung Cancer Provides a New Target for Cancer Therapy Pingfang Song 1 and Eliot R. Spindel 1, * 1 Division of Neuroscience, Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR 97006, USA Received September 8, 2007; Accepted December 11, 2007 Abstract. Lung cancer is the leading cause of cancer death worldwide and new treatment strategies are clearly needed. The recent discovery that lung and other cancers synthesize and secrete acetylcholine (ACh) which acts as an autocrine growth factor suggests that this cholinergic autocrine loop may present new therapeutic targets. In normal bronchial epithelium, small airway epithelium and pulmonary neuroendocrine cells synthesize Ach; and in squamous cell lung carcinoma, adenocarcinoma, and small cell lung carcinoma, the respective lung cancers that derive from those cell types similarly synthesize ACh. ACh secreted by those cancers stimulates growth of the tumors by binding to nicotinic and muscarinic receptors expressed on lung cancers. Thus antagonists to nicotinic and muscarinic receptors can inhibit lung cancer growth. The muscarinic receptor (machr) subtype utilized for cell proliferation is the M 3 subtype and consistent with this M 3 machr antagonists inhibit growth of SCLC and squamous cell carcinomas. This is significant as M 3 machr antagonists have low toxicity and are in wide clinical use. As multiple other cancer types besides lung carcinomas express both M 3 machr and acetylcholine, other cancer types besides lung carcinoma may respond to M 3 machr antagonists. Keywords: non-neuronal cholinergic system, lung cancer, cancer, non-neuronal acetylcholine, nicotinic receptor, muscarinic receptor Introduction Lung cancer is now the most common cause of cancer death worldwide and mortality numbers are still increasing in many countries due to changes in smoking frequency (1). Lung cancer survival remains poor with 5-year survival rates still below 20% for most lung cancers (2, 3); therefore, new therapeutic approaches to lung cancer are urgently needed. The recent discovery that lung cancers synthesize and secrete acetylcholine (ACh) that acts as a growth factor for lung cancers provides a potential new target to inhibit lung cancer growth (4). The concept of synthesis of ACh by nonneuronal tissue has been well established by the work *Corresponding author. spindele@ohsu.edu. Published online in J-STAGE doi: /jphs.FM of Kawashima et al. (5, 6), Wessler et al. (7, 8), and Grando et al. (9, 10), among others. Consistent with this, a variety of normal lung cell types synthesize ACh and the respective lung cancer types that arise from those cells similarly synthesize and secrete ACh (4, 11 13). The ACh secreted by lung cancers feeds back on the same cells and neighboring cells to stimulate growth in an autocrine or paracrine manner by binding to nicotinic acetylcholine receptors (nachr) and muscarinic acetylcholine receptors (machr) (4, 11). This suggests that lung cancer growth can potentially be targeted by blocking ACh synthesis, secretion, or signaling through nicotinic and muscarinic receptors. Of particular interest are pathways that might differentiate neuronal from non-neuronal cholinergic signaling. Thus a first step in analyzing the potential to target non-neuronal cholinergic signaling in lung cancer is to compare cholinergic signaling in lung to cholinergic signaling in neurons. 1

2 2 P Song and ER Spindel How does non-neuronal cholinergic signaling in lung differ from neuronal cholinergic signaling? The principal differences between cholinergic signaling in lung cells and neurons are outlined in Fig. 1. In neurons, the high affinity transporter CHT1 is absolutely required for ACh synthesis. CHT1 knockout mice fail to synthesize ACh and die at birth (14). By contrast, some lung cells such as pulmonary neuroendocrine cells make ACh but do not express CTH1. These cells may utilize a new class of choline transporters, the choline-transporter like proteins (CTL s) (15, 16). Lung cancers may similarly utilize the CTL s for choline transport as some lung cancers also synthesize ACh but do not express CHT1 (17). This is discussed further below. Synthesis of ACh by the enzyme choline acetyltransferase (ChAT) appears similar in both lung and neurons. Another key difference in ACh secretion between neurons and lung cells is ACh packaging and secretion. In neurons the ACh is packaged into vesicles by the actions of the vesicular ACh transporter (VAChT) and secretion of ACh in vesicles is a tightly regulated aspect of neuronal signaling and generated action potentials. In contrast, secretion of ACh by lung cells does not appear wholly dependent on VAChT, does not require action potentials, and is generally much less tightly regulated. Once ACh is released, it interacts with the same families of nachr and machr in both lung and neurons. Actions of ACh are terminated similarly in both lung and neurons by the cholinesterases AChE and BChE. In lung, the recently described ly6 proteins lynx1, slurp1, and slurp2 may play a role in modifying the activity of nachr (18, 19). In neurons, only lynx1 has been described to date (20). These differences are diagrammed in Fig. 1 and summarized in Table 1. Lung cancers express a cholinergic autocrine loop Just as normal lung cells express a cholinergic autocrine loop, lung cancers arising from the lung cells similarly express a cholinergic autocrine loop. Squamous cell carcinoma of the lung (SCC) arises from epithelial cells of bronchi and larger airways. Like bronchial epithelium, the majority of SCC express ChAT. In a series of 31 SCC examined in our laboratory 58% expressed ChAT (Fig. 2) (11). Bronchial epithelium expresses CHT1 (13), and consistent with that, the majority of SCC also express CHT1. How important CHT1 is for synthesis of ACh by SCC has not been established. Rounding out the autocrine loop, SCC express a variety of nicotinic receptors (21) and a high number also express the muscarinic M 3 receptor as discussed below (11). Adenocarcinomas have been less Fig. 1. Cholinergic signaling in neurons compared to signaling in lung. A: In neurons, choline is transported by the high affinity choline transporter CHT1, synthesized into ACh by choline acetyltransferase (ChAT), packaged into secretory vesicles by the vesicular acetylcholine transporter (VAChT), secreted in response to action potentials, and then interacts with nachr and machr in the synapse. Signaling is terminated by the action of acetylcholinesterase and butyrylcholinesterase. Signaling can also be modified by the action of the ly-6 protein lynx 1. B: By contrast in lung, CHT1 is not required for choline transport, the role of VAChT is unclear, secretion is less tightly regulated and not necessarily vesicular, and the ly-6 proteins Slurp1 and Slurp2 may also play a role. ChAT, nachr, machr, and the cholinesterases appear to function similarly in neurons and lung. studied, but in a series of bronchoalveolar lung cancers reviewed by us, 56% expressed ChAT (11) (Fig. 2). The cholinergic autocrine loop has been best characterized in small cell lung carcinoma, and the remainder of this review will focus on SCLC. In the series of SCLC reviewed by our laboratory, over 90% of SCLC express ChAT (Fig. 2) and essentially all expressed a number of nicotinic receptor subtypes, with alpha7 nachr expression being the most common. M 3 machr was expressed in 70% of SCLC (Fig. 3). In contrast to SCC, many SCLC do not express CHT1 and

3 Non-neuronal ACh and Lung Cancer 3 Table 1. Differences between neuronal and non-neuronal ACh synthesis Step Neuronal ACh synthesis Non-neuronal ACh synthesis Choline transport CHT1 only CHT1 and other transporters CHT1 required Yes No ACh synthesis c ChAT ChAT Vesicular storage and synthesis Yes Probably not VAChT required Yes Unknown Secretion regulated by action potentials Yes No Receptors nachr and machr nachr and machr Termination of signaling AChE and BChE AChE and BChE Allosteric modulators Lynx1 Lynx1, Slurp1, Slurp2 The differences between neuronal and non-neuronal ACh synthesis for each step of ACh synthesis are shown. Differences are shown in italics. Fig. 2. Expression of ChAT in lung cancers in squamous cell (A), bronchoalveolar (B), and small cell lung carcinoma (C) (chromogen = VIP, 400 ). Fig. 3. Co-expression of ChAT and M 3 machr in SCLC. A: ChAT immunostaining (400, chromogen = VIP), insert box = B: M 3R immunostaining (400, chromogen = VIP). C: Confocal image showing coexpression of M 3 machr (red) and ChAT (green) in tumor cells in the same sample as A and B. ACh synthesis does not appear dependent on CHT1. Instead, RT-PCR shows expression of multiple CTL subtypes in SCC, including essentially all subtypes (CTL1 5) (17). The cholinergic autocrine loop presents multiple targets to slow lung cancer growth How can the cholinergic autocrine loop be targeted to slow lung cancer growth? To study this, we have used

4 4 P Song and ER Spindel Fig. 5. The nicotinic agonist nicotine and muscarinic agonist carbachol both stimulate growth of H82 SCLC cells at the doses shown. *P<0.05, compared to the control by t-test. Fig. 4. SCLC cell lines synthesize ACh and express nachr and machr. A: ACh secreted into media by 500,000 cells of SCLC cell lines shown during 24 h in the presence of M neostigmine. B: Expression of nachr subunits in SCLC cell lines as shown by RT-PCR. C: Expression of machr subtypes in SCLC cell lines as shown by RT-PCR. Fig. 6. Cholinergic targets in lung cancer. The different points at which cholinergic signaling in lung cancer can be targeted are highlighted. Darker arrows showing choline uptake, ACh secretion, and the role of Lynx1 and the Slurps indicate potential differences between neuronal and non-neuronal ACh secretion. SCLC as a model system. As shown in Fig. 4, most SCLC cell lines express ACh, nachr, and machr. Consistent with ACh being a growth factor, both nicotinic and muscarinic agonists stimulate growth of the H82 SCLC cell line (Fig. 5). Conversely, the nicotinic antagonist mecamylamine and the muscarinic antagonist atropine both inhibit growth of H82 cells. In parallel with stimulation of H82 cell growth by either

5 Non-neuronal ACh and Lung Cancer 5 nicotine or carbachol, phosphorylation of MAPK and Akt is increased (11, 21). Knowing that cholinergic stimulation leads to cell proliferation through the MAPK and Akt pathways begins to suggest the multiple ways the cholinergic autocrine loop may be targeted to inhibit lung cancer growth. This is summarized in Fig. 6. Potential ways to target this pathway include blocking the nicotinic and muscarinic receptors with antagonists; modifying the actions of the ly6 proteins lynx1, slurp1, and slurp 2; modifying ACh secretion; modifying ACh synthesis; modifying choline uptake; and modifying the downstream proliferative pathways that involve MAPK and AKT. Of particular interest are pathways that may differ between non-neuronal cholinergic signaling and neuronal cholinergic signaling. This includes choline uptake, the secretory process itself (both mechanisms and regulation), and the potential roles of the ly6 proteins. M 3 receptor antagonists can slow growth of lung cancer A particularly attractive target in the cholinergic pathway is the M 3 muscarinic receptors. M 3 muscarinic receptor antagonists are in wide clinical use for overactive bladder (e.g., darifenacin and solifenacin) and COPD (tiotropium) and are generally well tolerated. This makes them a potentially interesting class of drugs to evaluate for use to slow cancer growth. As shown in Fig. 7A, M 3 antagonists block the ability of ACh to increase intracellular calcium. This in turn translates to blocking activation of Akt and MAPK and inhibition of cell growth (Fig. 7: B and C). This then translates into inhibition of growth in tumors by M 3 antagonists in nude mice in vivo (Fig. 8) Conclusions While the role of non-neuronal acetylcholine in lung has not yet been determined, it is clear that acetylcholine synthesized by cancers derived from the corresponding cell types acts as an autocrine and paracrine growth factor. Because the cholinergic signaling pathway is quite complex, this provides multiple targets for the inhibition of lung cancer growth. Particularly attractive is the M 3 muscarinic receptor that is widely expressed by most lung cancers as well as multiple other cancer types. While we show here that M 3 antagonists can inhibit growth of SCLC, they are also likely to inhibit growth of multiple other cancers as well that similarly express M 3 receptors. In summary, the cholinergic autocrine loop in lung cancers provides significant opportunities to develop new cancer therapies. Fig. 7. M 3 muscarinic receptor antagonists inhibit cholinergic signaling and resulting cell proliferation. A: The selective M 3 antagonist darifenacin inhibits the Ach-induced increase in intracellular calcium. Data are each presented as the mean ± S.E.M. B: Western blot showing that phosphorylation of Akt and MAPK induced by M ACh was decreased by the M 3 antagonist 4- DAMP in a concentration-dependent fashion. C: The M 3 machr antagonist 4-DAMP inhibited H82 cell proliferation in a concentration-dependent manner. Data are each expressed as the mean ± S.E.M. White column, control; dotted-pattern column, 10 9 M; diagonal-pattern column, 10 8 M; horizontal-pattern column, 10 7 M; grey column, 10 6 M; black column, 10 5 M. *P<0.001 and P<0.05, compared to the control at 9 days by the Tukey-Kramer multiple comparison test after 2-way ANOVA. References 1 Proctor RN. Tobacco and the global lung cancer epidemic. Nature Rev Cancer. 2002;1:82 86.

6 6 P Song and ER Spindel Fig. 8. Effect of darifenacin on growth of H82 tumor xenografts in nude mice. A: Tumor volume. *P<0.05, compared to the control at the same time point by the Tukey-Kramer multiple comparison test after repeated measures ANOVA. B: Tumor weight. *P<0.05, compared to the control by t-test. 2 Simon G, Ginsberg RJ, Ruckdeschel JC. Small-cell lung cancer. Chest Surg Clin N Am. 2001;11: Jackman DM, Johnson BE. Small-cell lung cancer. Lancet. 2005;366: Song P, Sekhon HS, Jia Y, Keller JA, Blusztajn JK, Mark GP, et al. Acetylcholine is synthesized by and acts as an autocrine growth factor for small cell lung carcinoma. Cancer Res. 2003;63: Kawashima K, Watanabe N, Oohata H, Fujimoto K, Suzuki T, Ishizaki Y, et al. Synthesis and release of acetylcholine by cultured bovine arterial endothelial cells. Neurosci Lett. 1990; 119: Kawashima K, Fujii T. Extraneuronal cholinergic system in lymphocytes. Pharmacol Ther. 2000;86: Wessler I, Kirkpatrick CJ, Racke K. Non-neuronal acetylcholine, a locally acting molecule, widely distributed in biological systems: expression and function in humans. Pharmacol Ther. 1998;77: Klapproth H, Reinheimer T, Metzen J, Munch M, Bittinger F, Kirkpatrick CJ, et al. Non-neuronal acetylcholine, a signalling molecule synthezised by surface cells of rat and man. Naunyn Schmiedebergs Arch Pharmacol. 1997;355: Zia S, Ndoye A, Nguyen VT, Grando SA. Nicotine enhances expression of the alpha 3, alpha 4, alpha 5, and alpha 7 nicotinic receptors modulating calcium metabolism and regulating adhesion and motility of respiratory epithelial cells. Res Commun Mol Pathol Pharmacol. 1997;97: Grando SA, Kist DA, Qi M, Dahl MV. Human keratinocytes synthesize, secrete, and degrade acetylcholine. J Invest Dermatol. 1993;101: Song P, Sekhon HS, Lu A, Arredondo J, Sauer D, Gravett C, et al. M3 muscarinic receptor antagonists inhibit small cell lung carcinoma growth and mitogenactivated protein kinase phosphorylation induced by acetylcholine secretion. Cancer Res. 2007;67: Song P, Sekhon HS, Proskocil B, Blusztajn JK, Mark GP, Spindel ER. Synthesis of acetylcholine by lung cancer. Life Sci. 2003;72: Proskocil BJ, Sekhon HS, Jia Y, Savchenko V, Blakely RD, Lindstrom J, et al. Acetylcholine is an autocrine or paracrine hormone synthesized and secreted by airway bronchial epithelial cells. Endocrinology. 2004;145: Ferguson SM, Bazalakova M, Savchenko V, Tapia JC, Wright J, Blakely RD. Lethal impairment of cholinergic neurotransmission in hemicholinium-3-sensitive choline transporter knockout mice. Proc Natl Acad Sci U S A. 2004;101: Fujita T, Shimada A, Okada N, Yamamoto A. Functional characterization of Na(+)-independent choline transport in primary cultures of neurons from mouse cerebral cortex. Neurosci Lett. 2005;393: Traiffort E, Ruat M, O Regan S, Meunier FM. Molecular characterization of the family of choline transporter-like proteins and their splice variants. J Neurochem. 2005;92: Song P, Spindel ER. Novel Na-independent choline transporters mediate choline transport and acetylcholine induced-proliferation in small cell lung carcinoma. Proc Am Thorac Soc (abstr). 18 Arredondo J, Chernyavsky AI, Grando SA. SLURP-1 and -2 in normal, immortalized and malignant oral keratinocytes. Life Sci In press. 19 Sekhon HS, Song P, Jia Y, Lindstrom J, Spindel ER. Expression of lynx1 in developing lung and its modulation by prenatal nicotine exposure. Cell Tissue Res. 2005;320: Miwa JM, Ibanez-Tallon I, Crabtree GW, Sanchez R, Sali A, Role LW, et al. Lynx1, an endogenous toxin-like modulator of nicotinic acetylcholine receptors in the mammalian CNS. Neuron. 1999;23: West KA, Brognard J, Clark AS, Linnoila IR, Yang X, Swain SM, et al. Rapid Akt activation by nicotine and a tobacco carcinogen modulates the phenotype of normal human airway epithelial cells. J Clin Invest. 2003;111:81 90.