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Cancer Research 67, 3936, April 15, 2007. doi: 10.1158/0008-5472.CAN-06-2484
© 2007 American Association for Cancer Research
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Endocrinology

M3 Muscarinic Receptor Antagonists Inhibit Small Cell Lung Carcinoma Growth and Mitogen-Activated Protein Kinase Phosphorylation Induced by Acetylcholine Secretion

Pingfang Song1, Harmanjatinder S. Sekhon1,2, Allison Lu1, Juan Arredondo4, David Sauer2, Courtney Gravett1, Gregory P. Mark3, Sergei A. Grando4 and Eliot R. Spindel1

1 Division of Neuroscience, Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, Oregon; Departments of 2 Pathology and 3 Behavioral Neuroscience, Oregon Health and Science University, Portland, Oregon; and 4 Department of Dermatology, University of California, Davis, California

Requests for reprints: Eliot Spindel, Division of Neuroscience, Oregon National Primate Research Center, Beaverton, OR 97006. Phone: 503-690-5512; Fax: 503-690-5384; E-mail: spindele{at}ohsu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The importance of acetylcholine as a neurotransmitter in the nervous system is well established, but little is yet known about its recently described role as an autocrine and paracrine hormone in a wide variety of nonneuronal cells. Consistent with the expression of acetylcholine in normal lung, small cell lung carcinoma (SCLC) synthesize and secrete acetylcholine, which acts as an autocrine growth factor through both nicotinic and muscarinic cholinergic mechanisms. The purpose of this study was to determine if interruption of autocrine muscarinic cholinergic signaling has potential to inhibit SCLC growth. Muscarinic receptor (mAChR) agonists caused concentration-dependent increases in intracellular calcium and mitogen-activated protein kinase (MAPK) and Akt phosphorylation in SCLC cell lines. The inhibitory potency of mAChR subtype–selective antagonists and small interfering RNAs (siRNAs) on acetylcholine-increased intracellular calcium and MAPK and Akt phosphorylation was consistent with mediation by M3 mAChR (M3R). Consistent with autocrine acetylcholine secretion stimulating MAPK and Akt phosphorylation, M3R antagonists and M3R siRNAs alone also caused a decrease in basal levels of MAPK and Akt phosphorylation in SCLC cell lines. Treatment of SCLC cells with M3R antagonists inhibited cell growth both in vitro and in vivo and also decreased MAPK phosphorylation in tumors in nude mice in vivo. Immunohistochemical staining of SCLC and additional cancer types showed frequent coexpression of acetylcholine and M3R. These findings suggest that M3R antagonists may be useful adjuvants for treatment of SCLC and, potentially, other cancers. [Cancer Res 2007;67(8):3936–44]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Annual global lung cancer deaths exceeded 1,000,000 in the year 2000 and are expected to exceed 2,000,000 by the year 2020 or 2030 (1). Lung cancers are classified as either small cell lung carcinoma (SCLC) or non-SCLC (NSCLC; ref. 2), with SCLC accounting for 15% to 20% of primary lung cancer (3, 4). NSCLC accounts for the remaining cases, with squamous cell and adenocarcinoma as the most common types of NSCLC (2). Current therapies for SCLC rarely extend survival beyond 5 years (3, 4); thus, novel therapeutic approaches for SCLC therapy are needed.

Acetylcholine is an important neurotransmitter in the central and peripheral nervous systems and plays key roles in learning, memory, autonomic control, and muscular contraction. Recently, it has been established that acetylcholine is also widely synthesized by a variety of nonneuronal cell types, including keratinocytes (5), airway epithelial cells (6, 7), glia (8), vascular endothelium (9, 10), placental trophoblasts (11), and ovarian follicular cells (12) among others (13). Expression of acetylcholine has been unambiguously shown in these cells and tissues as shown by the presence of choline acetyltransferase, the enzyme that synthesizes acetylcholine, and by demonstration of acetylcholine synthesis in these cells as measured by high-performance liquid chromatography (HPLC). The primary functions of nonneuronal acetylcholine have not yet been elucidated, but the expression of acetylcholine in many different cell types suggests that its roles will be fundamental and will present novel therapeutic targets. Our laboratory observation that pulmonary neuroendocrine cells synthesize acetylcholine led to our previous report that SCLC secrete acetylcholine, which acts as an autocrine growth factor for SCLC signaling through both nicotinic and muscarinic receptors (14). This new role for acetylcholine suggests a potential new pathway to target tumor growth in patients with SCLC.

Acetylcholine can stimulate cell growth through either nicotinic cholinergic or muscarinic cholinergic pathways. The muscarinic receptors (mAChR) are G-protein–coupled receptors, and five subtypes of mAChR (M1–M5) have been identified. The M1, M3, and M5 subtypes have been linked to cell proliferation and are coupled to Gq and upon activation lead to increased levels of intracellular IP3, diacylglycerol, and calcium ([Ca2+]I; ref. 15). The M2 and M4 receptors are coupled to Gi and inhibit adenylyl cyclase formation (15). Muscarinic stimulation of cancer growth has been reported for colon (16), lung (14), glial (17), and prostate cancers (18), and in ovarian carcinomas, expression of muscarinic receptors correlates with a poor prognosis (19). In colon and prostate carcinomas, tumor growth has been associated with stimulation of M3 receptors (16, 18).

The nicotinic cholinergic receptors (nAChR) are ligand-gated ion channels, and binding of acetylcholine allows entry of sodium or calcium into the cell. Nicotine is an exogenous ligand for nAChR, and multiple investigators have shown that activation of nAChR by nicotine stimulates lung cancer growth (20–24). Recently, West et al. have shown that nicotine stimulates tumor growth in NSCLC through AKT- and mitogen-activated protein kinase (MAPK)–dependent mechanisms (20), and that modulation of Akt signaling pathways may provide a target for directed therapy (25). Schuller et al. have also shown that the nitrosamine 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone (NNK) that is present in tobacco smoke similarly stimulates lung cancer growth through nicotinic receptors and MAPK-dependent mechanisms (23, 26). Although treatment with nicotinic receptor antagonists can have significant effects on blood pressure (27), muscarinic receptor antagonists are better tolerated and are widely used for treatment of chronic obstructive pulmonary disease (COPD; ref. 28) and overactive bladder (29). This suggests that muscarinic antagonists might serve as a useful adjuvant to conventional chemotherapeutic regimens for SCLC, which express the cholinergic autocrine loop.

Here, we show that the autocrine release of acetylcholine stimulates SCLC cell growth via M3 muscarinic mechanisms that involve increased [Ca2+]I and increased phosphorylation of MAPK. We also show that selective M3 antagonists can inhibit SCLC growth both in vitro and in vivo. In addition, because multiple other cancer types both synthesize acetylcholine and express M3 receptors, M3 antagonists may be an efficacious adjuvant therapy in many different oncologic protocols.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
SCLC cell culture and tissue samples. The classic SCLC cell lines NCI H69, H345, H592, and H1694 and the variant SCLC cell lines H82 and H417 were generously provided by A. Gazdar et al. (30) or obtained from the American Type Culture Collection (Rockville, MD) and maintained as previously described (14). Archival, paraffin-embedded, surgical, and bronchoscopic biopsies of lung and other cancers were obtained from the Department of Pathology, Oregon Health and Science University (OHSU), as approved by the OHSU Institutional Review Board for human studies.

Small interfering RNA transfection and assay. ON-TARGETplus small interfering RNAs (siRNAs) for the muscarinic receptor subtypes and negative control siRNA were purchased from Dharmacon (Lafayette, CO). The siRNAs were transfected at a concentration 100 nmol/L each with DharmaFECT 1 according to the manufacturer's instruction. Forty-eight hours after transfection, the cells were harvested for [Ca2+]I assay, Western blotting analysis, and preparation of total RNA. For cell proliferation assay, half the media was changed every 3 days for fresh media also containing the specific or negative control siRNAs and transfection reagent.

Western blot. Immunoblotting assay was used to detect M3 mAChR and the phosphorylation of p44/42 MAPK (extracellular signal-regulated kinase 1/2) and Akt elicited by exogenous and endogenous acetylcholine. For M3 mAChR, SCLC cells were lysed, and proteins were prepared as described previously (31). For MAPK and Akt phosphorylation, H82 cells in fresh media were treated by adding 3 x 10–5 mol/L acetylcholine for 5 to 120 min. For acetylcholine dose-response curves, 1 x 106 H82 cells in the fresh media were treated by adding 10–7 to 10–3 mol/L concentrations of acetylcholine. For the concentration-dependent effects of 4-DAMP on MAPK and Akt phosphorylation induced by exogenous acetylcholine, 1 x 106 H82 cells were preincubated in fresh media with 10–10 to 10–7 mol/L 4-DAMP for 30 min and then treated with 3 x 10–5 mol/L acetylcholine for 1 h. For the concentration-dependent effects of 4-DAMP on MAPK and Akt phosphorylation induced by endogenous acetylcholine, 2 x 106 H82 cells were incubated in fresh media 2 h and then treated with 10–11 to 10–8 mol/L 4-DAMP for 1 h. Cell pellets were washed with cold PBS and lysed by the addition of 50 to 100 µL SDS sample buffer [62.5 mmol/L Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mmol/L DTT, 0.01% bromophenol blue]. Samples were sonicated for 10 s, and 15 µL were loaded onto 10% SDS-PAGE gels. After electrophoresis, proteins were transferred onto 0.4-µm nitrocellulose membranes (Bio-Rad, Hercules, CA) and blocked with 5% nonfat dried milk in 0.05% Tween 20 (Sigma-Chemical Co., St. Louis, MO), 25 mmol/L Tris-HCl (pH 8), and 150 mmol/L NaCl. Rabbit antibodies against p44/42 MAPK, phosphorylated p44/p42 MAPK, Akt, phosphorylated Akt (Ser473; Cell Signaling Technology, Danvers, MA), and M3 mAChR (Research and Diagnostic Antibodies, North Las Vegas, NV) were diluted by 1,000- to 2,000-fold in the blocking buffer and incubated overnight with membranes at 4°C. Goat anti-rabbit immunoglobulin labeled with horseradish peroxidase (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was diluted 1:2,000 in the blocking buffer and incubated with the membrane for 1 h at room temperature. Membranes were developed using the ECL Plus chemiluminescent detection system (Amersham Pharmacia Biotech, Piscataway, NJ).

Acetylcholine assay. Acetylcholine secreted into media by SCLC cell lines was assayed by HPLC coupled to an enzyme-linked electrochemical detector as described previously (14). Aliquots of media were centrifuged, snap frozen, and stored at –80°C. For assay, 20-µL medium was injected directly into the HPLC. All samples were measured at least in duplicate.

Calcium fluorometry. Changes in intracellular calcium [Ca2+]I concentration elicited by cholinergic ligands in H82 were assayed using the FLEXstation Calcium Assay kit (Molecular Devices Corp., Sunnyvale, CA). In brief, cell suspensions were centrifuged (200 x g, 2 min), and cell pellets were resuspended in fresh medium at a cell density of 0.8 x 106 per mL; 100 µL of cell suspension was added to each well of poly-L-lysine–pretreated black wall plates, and an equal volume of the loading buffer from the Calcium Assay kit was added to each well. Plates were centrifuged at 200 x g for 4 min then incubated for 1 h at 37°C in 5% CO2, 95% room air to load cells with the dye. Fluorescent responses to ligand were measured using a FLEXstation (Molecular Devices) with excitation wavelength at 485 nm, emission wavelength at 525 nm, and emission cutoff of 515 nm. All drugs used were diluted to desired concentrations with HBSS supplemented by 20 mmol/L HEPES and 2 mmol/L CaCl2. For cholinergic agonist dose-response curves, acetylcholine and carbachol (10–7 to 10–4 mol/L) were applied, and fluorescence was monitored for 60 s. For antagonist studies, 4-DAMP (10–9 to 10–6 mol/L), atropine, mecamylamine, pirenzepine, darifenacin, and AFDX 116 (10–8 to 10–5 mol/L) were used. Antagonists were added 2 min before the addition of acetylcholine. Changes in [Ca2+]I were calculated as a percentage of the increased fluorescent signal produced by 10–4 mol/L cholinergic agonists for acetylcholine or carbachol dose-response curves, or by 3 µmol/L acetylcholine in the antagonist studies.

Cell proliferation assay. H82 and H1694 cells were used to determine which mAChR subtype mediated SCLC growth responses to endogenous acetylcholine secretion. The M3 mAChR–selective antagonists 4-DAMP and p-F-HHSiD, the M1 mAChR–selective antagonist pirenzepine, and the M2/M4 mAChR–selective antagonist AFDX-116 were used to identify the responsible muscarinic subtype. Cells were plated as described previously (14). Drugs at final concentrations of 10–9, 10–8, 10–7, 10–6, and 10–5 mol/L were added immediately after cell plating, and half the volume of media plus drugs were changed every 3 days. Cell density was monitored using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay as previously described (14) or by measuring the reduction of resazurin to resorufin using the Cell Titer-Blue assay (Promega Corp., Madison, WI). Cell growth is shown as relative to values at day 0.

Nude mice studies. For tumor xenograft growth studies, mice (nu/nu; Charles River Laboratories, Wilmington, MA) were injected with SCLC tumor cells (NCI-H82 cells) and treated with the M3 receptor antagonist darifenacin, and tumor growth was monitored. Darifenacin was generously provided by Novartis Pharma AG (Basel, Switzerland). Six- to 7-week-old nude mice were housed in a pathogen-free room in microisolators with autoclaved bedding and fed autoclaved rodent chow and water. H82 cells were grown as described above and resuspended in fresh RPMI 1640 + insulin, transferrin, and selenium at a concentration of 5 x 106 per 0.25 mL. Cell were then mixed with an equal volume of Matrigel (BD Biosciences, San Jose, CA) to give a final concentration of 5 x 106 per 0.5 mL, and 0.5 mL of this cell suspension was then injected s.c. into the right flank of each mouse. Tumors were allowed to grow for 1 week, and then drug administration was initiated and continued for the next 4 weeks. Darifenacin was dissolved in 50% DMSO/50% PBS and administered by s.c. implanted osmotic minipumps (Alzet model no. 2004) at doses of 0.3, 1.0, and 3.0 mg/kg/d. Control animals received minipumps filled with 50% DMSO/50% PBS. Tumor volume was determined weekly by measuring with calipers (volume = height x width x depth). The study was terminated after 4 weeks of drug administration. Ten animals were used per group. At sacrifice, tumors were removed and weighed, and blood was collected for measurement of plasma darifenacin concentration (32). Tumors were fixed for histologic examination, and portions were frozen for acetylcholine, RNA, and protein analyses.

Immunohistochemistry and immunocytochemistry. Paraffin-embedded sections (5 mm) of SCLC and other tumor types were cut from tissue blocks and stained with H&E to confirm diagnosis and tissue integrity. Serial sections were processed for immunohistochemistry as previously described (14). Antibodies used were mouse anti–choline acetyltransferase (mAb 305; Chemicon International, Inc., Temecula, CA; 1:400) and rabbit anti–M3 mAChR (H210; Santa Cruz Biotechnology; ref. 33). All analyses also included non-immune serum controls. Intensity of immunohistochemical staining was scored from 0 to 4+ (where 0, no staining; 1+, focal weak staining; 2+, focal strong staining or diffuse weak staining; 3+, diffuse medium staining; 4+, diffuse strong staining). Dual immunohistochemical staining was done using the same primary antibodies and donkey anti-mouse Alexa 488– and donkey anti-rabbit Alexa 594–labeled second antibodies from Molecular Probes, Inc. (Eugene, OR).

Statistical analysis. In vitro cell proliferation was analyzed by two-way ANOVA followed by the Tukey-Kramer multiple-comparison test using NCSS 2002 (Kaysville, UT) statistical software. Tumor growth in nude mice was analyzed by two-way ANOVA with repeated measures followed by Tukey-Kramer multiple-comparison tests. Final xenograft tumor weight was analyzed by Student's t test. Values for EC50 and IC50 in the Calcium fluorometry were calculated with SOFTmax Pro 4.2 (Molecular Devices).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SCLC cell lines express functional M3 muscarinic receptors and secrete acetylcholine. For mAChR to provide a therapeutic target to block autocrine cholinergic stimulation of SCLC growth, SCLC must express functional muscarinic receptors and secrete acetylcholine. SCLC cell lines H69, H82, H345, H417, H592, and H1694 were screened for expression of mAChR by PCR, and all cell lines expressed all five subtypes of mAChR (data not shown). Next, fluorometry was used to identify the muscarinic receptor subtype mediating acetylcholine-induced increases in intracellular calcium. As shown in Fig. 1A , acetylcholine and carbachol caused a concentration-dependent increase of [Ca2+]I in H82 cells, with EC50 of 0.47 and 2.78 µmol/L, respectively. As low as 10–7 mol/L acetylcholine induced 20% of maximal response in H82 cells; 10–7 mol/L of the nonselective muscarinic antagonist atropine completely blocked the response of H82 cells to 10–5.5 mol/L acetylcholine, showing that the effect of acetylcholine on [Ca2+]I was mediated by mAChR (Fig. 1B). As shown in Fig. 1C, 4-DAMP (a selective M3 mAChR antagonist), pirenzepine (a selective M1 mAChR antagonist), and ADFX 116 (a selective M2/M4 mAChR antagonist) each caused the concentration-dependent inhibition of the [Ca2+]I increase elicited by acetylcholine in H82 cells. The inhibitory potency order was 4-DAMP > pirenzepine > AFDX 116, consistent with mediation of the effects of acetylcholine on [Ca2+]I by the M3 mAChR. A second M3-selective mAChR antagonist (darifenacin) also completely inhibited the acetylcholine-induced increase in [Ca2+]I (data not shown). Similar results were obtained in a second SCLC cell line (H1694; data not shown). The role of M3 receptors in mediating the effect of acetylcholine on [Ca2+]I was confirmed by siRNA knockdown. Transfection of siRNAs against the M1, M3, and M5 muscarinic receptors all knocked down their target RNAs by >60% (data not shown), but only knockdown of the M3 mAChR RNA blocked the acetylcholine induced increase in [Ca2+]I (Fig. 1D). Western blot analysis confirmed the expression of M3 mAChR in H82 and H1694 cells showing a single band of 70 kDa as expected (ref. 31; data not shown).


Figure 1
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  		Figure 1. Calcium responses to cholinergic agonists and antagonists in H82 cells. Cells were plated and treated as described in Materials and Methods. A, acetylcholine (ACh; {blacksquare}) and carbachol ({blacktriangleup}) caused a concentration-dependent increase in [Ca2+]I in H82 cells. B, representative trace of the [Ca2+]I response of H82 cells to acetylcholine in the presence or absence of atropine. C, rank order potency of selective muscarinic antagonists to inhibit the [Ca2+]I increase elicited by acetylcholine in H82 cells. Antagonists tested were 4-DAMP ({blacksquare}; a selective M3 antagonist), pirenzepine ({blacktriangleup}; a selective M1 antagonist), and AFDX 116 (bullet; a selective M2/M4 antagonist). The rank order potency of these antagonists is most consistent with mediation by the M3 mAChR. D, siRNA knockdown of M3 mAChR blocked the acetylcholine induced increase in [Ca2+]I but control and M1 and M5 mAChR knockdowns had no effect. bullet, control siRNA; {blacksquare}, M1 siRNA; {blacktriangleup}, M3 siRNA; {diamondsuit}, M5 siRNA. Points, mean of at least 12 replicates from three separate experiments; bars, SE.

 
Completing the cholinergic autocrine loop, as we have previously reported, H82 cells synthesize and secrete acetylcholine (14). In addition, four of five other SCLC cell lines examined (H69, H345, H592, and H1694) synthesized acetylcholine with average secretion of 41.5 ± 1.5 pmol per 5 x 105 cells per 24 h. No acetylcholine secretion was detectable from the H417 SCLC cell line.

M3-selective mAChR antagonists inhibit SCLC cell proliferation in vitro. Next, the ability of M3 muscarinic antagonists to block growth of SCLC cell lines in vitro was determined. As shown in Fig. 2A and B , the selective M3 mAChR antagonists 4-DAMP and p-F-HHSiD both significantly inhibited H82 cell proliferation at 9 days, whereas the selective M1 antagonist pirenzepine and the M2/M4–selective antagonist AFDX-116 had no effects on cell proliferation (Fig. 2C and D). 4-DAMP showed a dose-dependent inhibition of cell growth, and consistent with the lower potency of p-F-HHSiD compared with 4-DAMP, p-F-HHSiD inhibited cell growth only at the highest concentration tested. The M3-selective antagonist darifenacin also showed significant inhibition of H82 cell growth at concentrations of 10–6 and 10–7 mol/L (data not shown). Similar results were also obtained for the inhibition of growth in the H1694 SCLC cell line by muscarinic antagonists (data not shown). Because the only source of ligand for the M3 receptor in these studies was endogenously released acetylcholine, this suggests that in SCLC cell lines, endogenous acetylcholine functions as an autocrine growth factor signaling in part through activation of M3 mAChR.


Figure 2
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  		Figure 2. Regulation of H82 cell proliferation by mAChR subtype antagonists. H82 cells were plated and treated as described in Materials and Methods. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay was used to detect H82 cell growth after treatment with 4-DAMP, P-F-HHSiD (M3 mAChR antagonists), AFDX-116 (M2/M4 mAChR antagonist), and pirenzepine (M1 mAChR antagonist). A, the M3 mAChR antagonist 4-DAMP inhibited H82 cell proliferation in a concentration-dependent manner. B, the less potent M3 mAChR antagonist P-F-HHSiD only significantly inhibited H82 cell proliferation at a concentration of 10–5 mol/L. C, the M1-selective mAChR antagonist pirenzepine had no significant effect on cell growth. D, the M2/M4–selective mAChR antagonist AFDX 116 had no significant effect on cell growth. Columns, mean of 24 replicates of two separate experiments; bars, SE. White column, control; dotted-pattern column, 10–9 mol/L; horizontal-pattern column, 10–8 mol/L; diagonal-pattern column, 10–7 mol/L; gray column, 10–6 mol/L; black column, 10–5 mol/L. *, P < 0.001; {dagger}, P < 0.05, compared with control at 9 d by Tukey-Kramer multiple-comparison test after a two-way ANOVA.

 
Exogenous and endogenous acetylcholine stimulate phosphorylation of MAPK and Akt in SCLC cell lines. The potential role of MAPK and Akt in mediating the effects of autocrine acetylcholine secretion on growth of H82 cells was evaluated by measuring acetylcholine-induced phosphorylation. As shown in Fig. 3A , acetylcholine induced a concentration-dependent phosphorylation of MAPK and Akt. Phosphorylation of Akt and MAPK induced by acetylcholine was most potently blocked by the selective M3 receptor antagonist 4-DAMP (Fig. 3B). M1 and M2/M4 antagonists were less potent in blocking phosphorylation (data not shown). Thus, acetylcholine-induced phosphorylation of MAPK and Akt is mediated in part by M3 mAChR. However, if autocrine acetylcholine secretion regulates basal phosphorylation of MAPK and Akt, then addition of M3 antagonists alone should decrease MAPK and Akt phosphorylation. As shown in Fig. 3C and D, addition of 4-DAMP to H82 cells inhibited MAPK and Akt phosphorylation in a dose-wise manner, indicating that autocrine acetylcholine secretion regulates basal phosphorylation of MAPK and Akt in SCLC. Effects were more pronounced on MAPK than AKT. Similar results were also seen with H1649 cells (data not shown). Specific knockdown of M3 mACh RNA with siRNAs similarly blocked basal phosphorylation of MAPK and lead to decreased cell proliferation (Fig. 3E), thus confirming the role of M3 receptors.


Figure 3
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  		Figure 3. Effect of acetylcholine on phosphorylation of MAPK and Akt in H82 SCLC cells. A, Western blot showing increased MAPK and Akt phosphorylation induced by concentrations of acetylcholine shown. B, Western blot showing that phosphorylation of Akt and MAPK induced by 3 x 10–5 mol/L acetylcholine was decreased by the M3 antagonist 4-DAMP in a concentration-dependent fashion. C, Western blot showing that 4-DAMP alone decreased basal phosphorylation of Akt and MAPK. D, quantitation of MAPK and Akt phosphorylation shows the dose-wise inhibition induced by 4-DAMP alone. E, effect of siRNA knockdown of the M3 mAChR on phosphorylation of MAPK. Left, representative MAPK Western blot; middle, quantitation of the decrease in MAPK phosphorylation caused by M3 receptor knockdown; right, result of the M3 receptor knockdown on cell proliferation. Points/columns, mean of two independent experiments; bars, SE. *, P < 0.005.

 
The M3 receptor antagonist darifenacin inhibits SCLC xenograft growth and MAPK phosphorylation in vivo in nude mice. If autocrine acetylcholine secretion has a regulatory role on tumor growth, then M3 antagonists should inhibit tumor growth in nude mice xenografts. As shown in Fig. 4A and B , consistent with the effects of M3 receptor antagonists in vitro, the M3-selective receptor darifenacin significantly inhibited H82 tumor growth in a dose-wise manner. Darifenacin was used because of its in vivo stability and high degree of M3 selectivity. No significant effects of darifenacin treatment on overall animal weight were observed. As shown in Fig. 4C, significant inhibition of tumor growth were achieved at a plasma concentration of 9.4 ± 0.3 ng/mL (~2.5 x 10–8 mol/L), which is a concentration achieved with clinical use of darifenacin for overactive bladder (32).


Figure 4
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  		Figure 4. Effect of darifenacin on growth of H82 tumor xenografts in nude mice. A, tumor volume. *, P < 0.05, compared with control at same time point by Tukey-Kramer multiple-comparison test after repeated-measures ANOVA. B, tumor weight. *, P < 0.05, compared with control by t test. C, plasma darifenacin concentrations obtained in nude mice receiving darifenacin doses as shown. Dashed and dotted lines, concentration of darifenacin achieved in human patients taking 30 and 15 mg darifenacin once per day, respectively (32). Columns, mean of 8 to 10 animals; bars, SE. 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 along with representative bands from Western blots for each treatment: N = 8 for 3.0 mg/kg/d and control doses and N = 4 for other doses. The lower doses of darifenacin also had no effect on Akt phosphorylation (data not shown). *, P < 0.05, compared with control by t test.

 
Consistent with the in vitro effects of M3 antagonists, levels of MAPK phosphorylation in the tumors removed from the darifenacin treated mice were significantly decreased (Fig. 4D), whereas no significant effects were seen on Akt phosphorylation (Fig. 4D). Acetylcholine content of control tumor samples was 420 ± 110 pmol/g, which corresponds to 4 x 10–7 mol/L, assuming uniform distribution in the tumor. Based on the responses shown in Fig. 3, these levels of acetylcholine are high enough to increase MAPK phosphorylation. Histologically, cells in tumors from nude mice treated with the highest dose of darifenacin were larger, with increased cytoplasm and less nuclear molding compared with controls consistent with slower proliferation.

Coexpression of choline acetyltransferase and M3 mAChR subtype in SCLC and other cancers. The above data show that in SCLC cell lines that both synthesize acetylcholine and express M3 receptors, M3 receptor antagonists and siRNA can inhibit cell proliferation. To determine the frequency of SCLC that both synthesize acetylcholine and express M3 receptors, a panel of archival SCLC specimens was screened for coexpression of M3 mAChR and choline acetyltransferase, the enzyme necessary for acetylcholine synthesis. In a series of 24 SCLC tumors, 17 of 24 (70%) tumors expressed M3 receptor immunoreactivity with an average intensity of 1.2 of 4 as described in Materials and Methods (Table 1 ). In addition, all 17 of the SCLC that expressed M3 also expressed choline acetyltransferase, with average immunostaining intensity of 1.4 of 4 (Table 1). Thus, 70% of the SCLC screened expressed both choline acetyltransferase and M3R, suggesting that M3 receptor antagonists could inhibit the growth of the majority of SCLC. Representative staining of choline acetyltransferase and M3 in SCLC is shown in Fig. 5 . As shown in Fig. 5C, individual tumor cells coexpress M3R and choline acetyltransferase. Specificity of staining is shown in Fig. 5D, which shows an example of a SCLC tumor that did not express choline acetyltransferase.


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  		Table 1. Frequency of choline acetyltransferase and M3 coexpression in selected cancers

 

Figure 5
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  		Figure 5. Immunohistochemistry of choline acetyltransferase (ChAT) and M3 mAChR expression in an SCLC biopsy. A, choline acetyltransferase immunostaining (x400, chromogen = VIP). Inset, x1,000. B, M3R immunostaining (x400, chromogen = VIP). C, confocal image showing coexpression of M3 mAChR (red) and choline acetyltransferase (green) in tumor cells in same sample as (A) and (B). D, example of negative staining for choline acetyltransferase in an SCLC tumor (Tumor) in which the expected positive staining for choline acetyltransferase in the normal airway epithelium (Epi) also on the section can be seen (x200).

 
Previous studies from our laboratory and other laboratories have shown that acetylcholine and M3 receptors are expressed in a wide variety of normal endothelial and epithelial cell types (5–13). This suggests that cancers derived from these diverse cell types will similarly coexpress acetylcholine and M3 receptors. As shown in Table 1, this is the case for NSCLC in which coexpression of choline acetyltransferase and M3 receptors occurs in 70% of bronchoalveolar lung carcinoma and 45% of squamous cell carcinoma of the lung. Coexpression of acetylcholine and M3 receptors is not limited to lung cancers, as 50% of pancreatic carcinoma and 43% of cervical carcinoma also show such coexpression (Table 1).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Autocrine and paracrine secretion of acetylcholine by nonneuronal cells represents a new paradigm for the physiologic and pathologic functions of acetylcholine. The role of acetylcholine as a neurotransmitter in the central and peripheral nervous system is a bedrock of neuroscience, but essentially nothing is known of the role of nonneuronal acetylcholine and the therapeutic opportunities this new pathway may provide. Currently, there is intense research on inhibition of proliferative tyrosine kinases in the treatment of many cancers, and blocking autocrine cholinergic signaling may provide a directed upstream approach to blocking proliferative pathway phosphorylation. In this study, we have now shown that autocrine secretion of acetylcholine by SCLC plays a role in maintaining basal phosphorylation of Akt and MAPK, and that widely prescribed M3 mAChR antagonists (28, 29) can decrease Akt and MAPK phosphorylation and concomitantly decrease tumor growth.

Evaluation by PCR showed that all five muscarinic receptor subtypes are present in all SCLC cell lines we have examined to date. Studies in diverse cell types have shown that muscarinic receptor activation is associated with cell proliferation, and this seems to be linked to increases in intracellular calcium mediated by the M1, M3, and M5 receptors, which are linked to Gq. In SCLC, Fucile et al. (34) and Beekman et al. (35) have shown the importance of Gq- and calcium-linked signaling in stimulation of tumor growth. Consistent with this, muscarinic agonists increased [Ca2+]I in SCLC (Fig. 1). Antagonist studies suggested this increase in [Ca2+]I was mediated by M3 muscarinic receptors, and this finding was confirmed using three different M3 antagonists. In addition, siRNA was used to confirm the role of the M3 receptor. Knockdown of the M1 and M5 mAChR receptors had no effect on the acetylcholine-induced increase in [Ca2+]I, whereas knockdown of the M3 receptor almost completely blocked the calcium response.

For the cholinergic autocrine loop to be present, cells must also express acetylcholine, the ligand for the M3 mAChR. Five of six SCLC cell lines examined here synthesized and secreted acetylcholine. This is consistent with the reports that acetylcholine is synthesized and secreted by nonneuronal cell types, including airway epithelial cells (7, 36), vessel endothelial cells (37), lymphocytes (38), ovarian granulosa cells (39), and placenta (40). During a 24-h incubation, >100 pmol of acetylcholine were secreted by 5 x 105 H82 cells, and the acetylcholine concentration in the medium reached 150 nmol/L, a concentration sufficient to trigger [Ca2+]I increase in SCLC cells (Fig. 1). In the local environment of tumors, concentrations of acetylcholine at surface receptors may be even higher due to high cell densities in solid masses, proximity of secretion events to receptor location, and variations in cholinesterase levels. It has also been reported that levels of circulating cholinesterase is reduced in cancer patients (41), and decreased levels of cholinesterase in NSCLC has been reported by Martinez-Moreno et al. (42), with the implication that cholinergic signaling may be further increased in tumors due to decreased cholinesterase activity.

If acetylcholine secreted by SCLC stimulates tumor growth through M3 muscarinic mechanisms, the addition of M3 mAChR antagonists should inhibit SCLC growth. As shown in Fig. 2, this is so, and the M3-selective antagonists 4-DAMP, p-F-HHSiD, and darifenacin significantly inhibited SCLC growth in vitro, whereas the M1 antagonist pirenzepine and the M2/M4 antagonist AFDX-116 had no significant effect. As shown in Fig. 3, the M3 mAChR antagonists also inhibited acetylcholine-induced phosphorylation of MAPK and Akt in H82 cells. As for inhibition of cell proliferation, the rank order potency of mAChR antagonists to block MAPK and Akt phosphorylation by acetylcholine in H82 cells was 4-DAMP > pirenzepine > AFDX 116. This suggests that M3 receptors may mediate the effects of autocrine acetylcholine on cell proliferation through stimulation of phosphorylation of MAPK and Akt. Knockdown of the M3 receptor with siRNAs confirmed this, causing similar decreases in both basal phosphorylation of MAPK and SCLC growth in vitro (Fig. 3E) as did the selective M3 antagonists.

These findings are consistent with studies of muscarinic stimulation of tumor growth in multiple cancer types. Fucile et al. (34) have similarly reported muscarinic stimulation of SCLC cell growth. M3 receptors have also been shown to mediate growth in prostate carcinoma cell lines (18) and gastric carcinoma cell lines (43). Frucht et al. (44), Cheng et al. (16), and Ukegawa et al. (45) have all shown that acetylcholine stimulates proliferation of colon carcinoma cell lines through M3-dependent phosphorylation of MAPK pathways. In astrocytoma cells, M3 receptors also stimulate growth through the Akt and MAPK pathways (46, 47), and cholinergic stimulation stimulates proliferation of breast cancer cells through MAPK (48). Acetylcholine expression has been shown in the ovary (39), and the expression of muscarinic receptors in ovarian cancer correlates with decreased survival (19). Thus, these studies are in concordance with our finding that acetylcholine stimulates lung cancer growth through M3 mAChR linked to MAPK phosphorylation.

These findings raise the question whether autocrine acetylcholine secretion from lung cancer cells plays a role in maintaining basal phosphorylation of MAPK and Akt in tumor cells. This seems to be the case because as shown in Fig. 3C and D, the M3 antagonist 4-DAMP decreases basal phosphorylation of Akt and MAPK in H82 cells in a dose-dependent manner. Of note, West et al. have shown that the interaction of nicotine with nicotinic receptors also leads to increased Akt (20); thus, acetylcholine secreted by lung tumors may also stimulate Akt and MAPK phosphorylation through interaction with nicotinic receptors. Other paracrine sources of acetylcholine, such as airway epithelium (6, 7), may also be important especially in smaller tumors. Indeed, Reinheimer et al. (6) has measured acetylcholine content of human bronchi of 2,500 pmol/g, which assuming a uniform distribution, would correspond to a concentration of 2.5 x 10–6 mol/L. Based on the data shown in Figs. 1 and 3, this would be enough to increase intracellular calcium and stimulate MAPK phosphorylation. The implication of this is that in smokers with lung cancer, cholinergic-mediated proliferative pathways will be stimulated both by exogenous nicotine and from endogenous acetylcholine. Furthermore, as nicotine has been shown to up-regulate levels of its receptor (49), the interaction between exogenous and endogenous cholinergic stimulation is likely to be synergistic with significant potential to lead to cellular hyperplasia/proliferation and phenotypic progression (20, 50). Finally, Schuller et al. (23, 26) have shown that the carcinogen NNK present in cigarette smoke also signals through nicotinic pathways to activate MAPK in lung cancers to further synergize with endogenous cholinergic signaling.

An important finding of this study is that M3 mAChR antagonists inhibit SCLC growth in vivo. As shown in Fig. 4, growth of the SCLC H82 cell line was inhibited by the M3 antagonist darifenacin in xenografts in nude mice. This confirms that tumor secretion of acetylcholine in vivo stimulates SCLC tumor growth, and that M3 receptor antagonists can block autocrine cholinergic stimulation of tumor growth. As shown in Fig. 4C, inhibition of tumor growth was achieved at plasma darifenacin concentrations between 3 and 10 ng/mL, which are in the range of plasma concentration obtained with clinical use of darifenacin (32). Thus, inhibition of SCLC growth can be achieved at clinically relevant doses of M3 receptor antagonists.

As shown in Fig. 4, inhibition of tumor growth in vivo was associated with inhibition of MAPK phosphorylation within the tumor, whereas Akt phosphorylation was not inhibited. This suggests that in vivo, autocrine acetylcholine signals proliferation primarily through the MAPK pathway, and that there are other factors within serum or the tumor that stimulate Akt phosphorylation. This suggests that a combination regimen of Akt inhibitors and M3 receptor antagonists may be effective in inhibiting tumor growth, especially in smokers in whom nicotine has been shown to activate Akt proliferative pathways (20, 50).

As discussed above, activation of M3 mAChR stimulates growth in multiple tumor types. Thus, if the many tumors that express M3 receptors also synthesize acetylcholine, the cholinergic autocrine loop may be wide spread in cancers. In our study, screening of archival tumor samples showed that expression of the M3 muscarinic cholinergic autocrine loop is widespread in SCLC. Seventeen of 24 SCLC examined expressed M3 receptors, and all tumors that expressed M3 receptors also expressed choline acetyltransferase. In addition, the widespread expression of acetylcholine and M3 receptors in nonneuronal cells suggests the autocrine cholinergic loop may occur in other tumor types besides SCLC. This is supported by the data shown in Table 1, with significant expression of choline acetyltransferase and M3 receptors in squamous cell lung carcinoma, bronchoalveolar lung carcinoma, and in two non-lung carcinoma types as well. Although we have not looked at the proliferative responses of cancer types other than SCLC to acetylcholine, data on the effects of M3 activation on growth of a variety of cancers types (16, 18, 43–48) suggest that M3 receptor antagonists will also inhibit growth of other cancer types that similarly both synthesize acetylcholine and express M3 receptors. Identification of which cancer types can be inhibited by M3 receptor antagonists and what criteria are necessary to identify the subset of tumors that will respond to M3 antagonists remains to be determined.

M3 receptor antagonists are widely used for treatment of COPD and overactive bladder. Thus, clinical trials to determine the usefulness of M3 antagonists as adjuvants to existing cancer therapy for SCLC and other cancer types may be warranted. Although M3 mAChR antagonists are unlikely to be a primary therapy, in combination with existing therapies they may have potential to slow tumor growth with minimal side effects.


   Acknowledgments
 
Grant support: NIH grants RR-00163 and HD/HL-37131.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Yibing Jia for technical assistance with real-time PCR, Kalama Taylor and CoreyAyne Singleton for technical assistance with cell culture, Anda Cornea for technical assistance with confocal microscopy, and Hans-Juergen Pfannkuche and Slavica Milosavljev from Novartis Pharma for providing darifenacin and measuring the levels of darifenacin in mouse plasma.

Received 7/ 7/06. Revised 12/11/06. Accepted 2/ 9/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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