Tumor-suppressor function of muscarinic acetylcholine receptors is ...

Proc. Natl. Acad. Sci. USA Vol. 90, pp. 1706-1710, March 1993 Medical Sciences

Tumor-suppressor function of muscarinic acetylcholine receptors is associated with activation of receptor-operated calcium influx (signal transduction/tyrosine kinase/CHO ceU/phospholipase A2/phospholipase D) CHRISTIAN C. FELDER*t, LINDA MACARTHUR*, ALICE L. MA*, FABIAN GUSOVSKYt,

ELISE C. KOHN§ Medicine Branch and Laboratory of Pathology, National Cancer Institute, and *Laboratory AND

*Laboratory of Cell Biology, National Institute of Mental Health, of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892

Communicated by Julius Axelrod, October 6, 1992

receptor subtypes in tumorigenic CHO-Kl cells offered a model from which to study ligand-driven inhibition of tumorigenesis and to determine the role of different signal transduction pathways in this process. We now report mus-

Several members of the family of guanine ABSTRACT nucleotide-binding protein (G protein)-coupled receptors have recently been shown to induce agonist-dependent foci development in NIH 3T3 cells and tumors in nude mice. We selected the five subtypes of the muscarinic acetylcholine receptor family to investigate their role in tumor suppression. When transfected and expressed in CHO-Kl Chinese hamster ovary cells, ml, m3, and m5 muscarinic acetylcholine receptor activation resulted in a morphology change. Receptor activation did not slow or inhibit monolayer growth of CHOm5 cells in culture but markedly inhibited density-independent growth in soft agar and suppressed tumor formation in nude mice. Receptor-mediated tumor suppression was found to be agonistdependent and reversible and was blocked with a muscarinic receptor antagonist. Of the five signaling pathways associated with the ml, m3, and m5 receptors, only receptor-operated, and inositol trisphosphate-independent, calcium influx was found to correlate with inhibition of tumorigenicity. These data suggest a pivotal role for inositol trisphosphate-independent receptor-regulated calcium homeostasis in CHO-Kl tumor suppression.

carinic receptor-mediated suppression of density-independent growth and tumor formation in nude mice and explore underlying receptor-driven mechanisms of that tumor suppression.

EXPERIMENTAL PROCEDURES Materials. [5,6,8,9,11,12,14,15-3H(N)]Arachidonic acid, [9,10-3H(N)]palmitic acid, and [3H]inositol were purchased from American Radiolabeled Chemicals (St. Louis). Reagents for the radioimmunoassay of cAMP were purchased from Gary Brooker (Georgetown University, Washington, DC). Anti-phosphotyrosine antibody was purchased from Upstate Biotechnology (Lake Placid, NY). All other reagents were purchased from Sigma. Cell Culture and Transfection. CHO-Kl cells were obtained from the American Type Culture Collection and maintained as described (9). CHO-Kl cells stably expressing the five subtypes of the rat muscarinic receptor were gifts of Tom Bonner, Noel Buckley, and Mark Brann (13). A clonal CHO-Kl cell expressing the m5 receptor with high carbacholinduced transformation potential was selected by limiting dilution and was used in all experiments. CHO cell morphology was measured as described (12). Soft-Agar Colonization. Soft-agar colonization and thymidine uptake of CHOmS cells were assessed as described (14). Tumor Formation. Female BALB/c nude (nu/nu) mice were inoculated subcutaneously with 106 CHOm5 cells per site at four sites per mouse, one mouse per three experiments. Inoculation sites were treated with 20 /4 of dimethyl sulfoxide vehicle, carbachol (1 mM), or carbachol plus atropine (1 mM) twice daily for the duration of the study. Average mass of the spherical tumors was determined by measuring their diameter and converting to millimeters cubed. Only measurable lesions were included in the calculations; unmeasurable sites were not included in the denominator to avoid bias in reporting. Data are the mean ± SE of all 12 inoculation sites. Radioligand Binding Assays and Assays of Muscarinic Receptor-Stimulated Second Messengers. Plasma membrane preparation and radioligand binding assays were performed (13) with 4-diphenylacetoxy-N-(2-chloroethyl)piperidine as the labeled ligand and 50 ,aM atropine to define nonspecific binding. Binding data were analyzed with the LIGAND pro-

Transmembrane receptors serve as a means for cells to recognize and integrate external signals and also play an important role in both regulated and aberrantly regulated cellular proliferation. Unlike the growth factor receptors, the guanine nucleotide-binding protein (G protein)-coupled class of receptors do not contain tyrosine kinase activity. These receptors, which have as their prototype the bacteriorhodopsin receptor, have seven transmembrane domain structures in common (1). The third cytoplasmic loop of these receptors confers specificity for G-protein coupling. These receptors couple to a diverse group of effector mechanisms, including membrane-associated phospholipases, adenylate and guanylate cyclases, and ion channels. Although these receptors are different from the growth factor receptors in that they bind to G proteins and lack the intrinsic kinase activity, in some cases similar second-messenger systems are activated (2-4). Expression of several members of this family of serpentine receptors in mouse NIH 3T3 cells permits agonist-dependent foci development in culture and tumor formation when the cells are inoculated into nude mice (5-7). The potential role for these receptors in cells already predisposed to tumor formation has not yet been analyzed. The five subtypes of muscarinic acetylcholine receptors (mil-mS) constitute a well-characterized example of G protein-coupled receptors (8-12). The odd-numbered receptors couple to an array of effector enzymes and ion channels, whereas the evennumbered receptors are coupled to Gi and inhibit adenylate cyclase activity. Transfection and stable expression of all five

Abbreviations: IP3, inositol 1,4,5-trisphosphate; CAI, a substituted carboxyamidotriazole (L651582, NSC 609974); BABA,-2-hydroxy5-(2,5-dihydroxybenzyl)aminobenzoic acid; RCAM, reduced carbamoylmethylated. tTo whom reprint requests should be addressed at: Laboratory of Cell Biology, Building 36, Room 3A-15, National Institute of Mental Health, Bethesda, MD 20892.

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Medical Sciences: Felder et al. gram (15) or with the GRAPHPAD program (GraphPad Software, San Diego). Methods for the determination of secondmessenger generation have been described for [3H]arachidonic acid release (10), inositol 1,4,5-trisphosphate (IP3) release (11), phosphatidylethanol release (16), cAMP accumulation (11), and 45Ca2+ uptake (11). Tyrosine Phosphorylation in CHOm5 CeUls. Serum-starved confluent CHO cells were pretreated with phosphate-free Dulbecco's modified Eagle's medium containing 1% dialyzed fetal bovine serum and 0.5 mCi (18.5 MBq) of carrier-free [32P]orthophosphate for 4 hr at 37°C. Incubation buffer (20 mM Hepes/4.7 mM KC1/0.5 mM EDTA/118 mM NaCl/5.0 mM NaHCO3/1.2 mM MgSO4/1.2 mM KH2PO4/2.5 mM CaCI2, pH 7.4) containing experimental agents was added and the 15-min reaction was stopped with three washes of phosphate-buffered saline containing 1.0 mM Na3VO4, followed by 0.5 ml of lysis buffer [50 mM Hepes/50 mM NaCl/50 mM NaF/5.0 mM EDTA/1.0 mM Na3VO4/1% (vol/vol) Nonidet P-40/1.0 mM phenylmethanesulfonyl fluoride with aprotinin and leupeptin at 10 ,ug/ml]. After 30 min at 4°C, the reaction mixture was centrifuged at 12,000 x g and the supernatant was incubated with 5-10 ,ug of anti-phosphotyrosine antibody for 2 hr at 4°C. Thirty microliters of preswollen protein A-Sepharose CL-4B was added and incubated 45 min at 4°C. After brief centrifugation, the pellet was washed four times with phosphate-buffered saline, SDS/PAGE loading buffer was added, and the sample was boiled for 3 min. Following SDS/10% PAGE, the gel was dried and exposed to x-ray film for 24 hr.

RESULTS Muscariliic Receptor-Dependent Suppression of Tumorigenicity. Under normal growth conditions, parental CHO-Kl cells have a stellate morphology (Fig. 1) and form tumors when inoculated into nude mice (Fig. 1H). When stimulated with the -muscarinic receptor agonist carbachol, CHO-Kl cells, stably expressing either the ml, m3, or m5 muscarinic receptor (CHOml, -m3, or -m5), changed shape to a bipolar, fibroblast-like morphology (Fig. 1 B, D, and F); this change was blocked by the addition of the muscarinic receptor antagonist atropine (Fig. 1G). The response was fully reversed following removal of agonist and did not occur in A /A

Proc. Natl. Acad. Sci. USA 90 (1993)

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CHOm2, CHOm4, or sham-transfected cells (Fig. 1 A, C, and E). Thymidine incorporation was unchanged following carbachol addition, suggesting that the morphology change was not the result of growth inhibition (data not shown). m5 receptor-mediated suppression of the tumorigenic phenotype was first evaluated by colony formation in soft agar. Untreated CHOmS cells initially formed discrete colonies that grew and then spread until individual colonies merged (Fig. 2A). In contrast, carbachol-treated CHOmS cells formed fewer and smaller discrete colonies, which failed to grow or spread and were reversed with the addition of atropine (Fig. 2B). Atropine had no effect on its own (data not shown). These results are consistent with a m5 muscarinic receptor-mediated reduction in the ability of CHOm5 cells to display density-independent growth in soft agar. The tumorigenic potential of carbachol-treated CHOm5 cells was further evaluated in a nude mouse model (Fig. 3). CHOm5 cells were injected at four subcutaneous sites at a density of 106 cells per site, and each site was bathed twice daily with vehicle or experimental agents. A rapid onset and growth of tumors was observed in animals receiving dimethyl sulfoxide vehicle following CHOmS cell injection. In contrast, carbachol treatment resulted in a marked delay in the time of appearance of tumors (Fig. 3). A 100% incidence of tumor formation was seen for mice receiving vehicle or carbachol plus atropine, whereas carbachol treatment caused 75% reduction in tumor incidence. Discontinuation of carbachol treatments resulted in a rapid appearance and growth of tumors (data not shown). These results indicate that m5 receptor activation was required to reduce the tumorigenic potential of the CHOmS cells in nude mice. A fibrosarcoma with a high mitotic rate, lack of cellular organization, and areas of necrosis was seen on histologic review of the CHOm5 cell-induced tumor mass (Fig. 1H). Radioligand binding experiments demonstrated that both receptor affinity and density binding parameters were identical in membranes derived from untreated CHOmS cells or from cells cultured from the vehicle-treated mouse CHOm5 tumors (CHOm5 parental cells; Kd = 0.12 + 0.02 nM, B..,, = 2.9 ± 0.2 pM; tumor-derived cells, Kd = 0.09 + 0.01 nM, Bmax = 3.3 + 0.1 pM). Correlation of Muscarinic Receptor-Stimulated Signal Transduction with CHOm5 Cell Morphology Change. Previ-

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B FIG. 1. Muscarinic subtype specificity for induction of a morphology change of CHO-Kl cells. (A) Sham-transfected CHO-Kl cells. (B) CHOml cells plus 10 AM carbachol. (C) CHOm2 cells plus 10 ,uM carbachol. (D) CHOm3 cells plus 10 ,uM carbachol. (E) CHOm4 cells plus 10 ,uM carbachol. (F) CHOmS cells plus 10 ,uM carbachol. (G) CHOmS cells plus 10 ,uM carbachol and 10 ,AM atropine (A-G: differential interference contrast microscopy; x300). (H) Histology of CHOmS tumor formed at the nude mouse inoculation site (hematoxylin/eosin; x200.)

Medical Sciences: Felder et al.

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studies have demonstrated multiple signal transduction pathways associated with the m5 receptor when expressed in CHO-Kl cells. These include activation of phospholipase A2, phospholipase C, adenylate cyclase, and 0P3-independent receptor-operated calcium channels (9-12, 17). The present study includes evidence for m5 activation of phospholipase D in CHO-Kl cells. Dose-response curves for carbachol stim-

ous

Proc. Natl. Acad Sci. USA 90 (1993)

ulation were generated for each of the signal transduction pathways and were compared with the dose-response curve for the carbachol-stimulated morphology change as measured by change in length/width ratios (Fig. 4). Arachidonic acid, IP3, phosphatidylethanol, and cAMP generation were used to measure phospholipase A2, C, D, and adenylate cyclase activation, respectively. The EC50 for carbacholstimulated morphology change was similar to that for muscarinic receptor-activated calcium influx, but both were 2 orders of magnitude more sensitive than the doses of carbachol required to activate the other signal transduction pathways (Table 1). The CHOmS morphology change required chronic carbachol treatment for 6 hr or more for full development. m5 receptor-stimulated calcium transients associated with IP3 production did not persist beyond 5 min, yet the sustained calcium influx was observed for >2 hr (data not shown). The correlation of sensitivity and temporal persistence of IP3-independent calcium influx with receptorstimulated morphology changes in response to carbachol suggested independence from the other pathways tested. The dependency of CHOm5 morphology change on receptor-activated calcium influx was further substantiated when the calcium channel blocker CAI (11), a substituted carboxyamidotriazole (L651582, NSC 609974), blocked agonistdependent elongation (Fig. 5). We showed previously that CAI had little effect on m5 receptor-stimulated inositol phosphate release or cAMP formation and that CAI blocked carbachol-stimulated 45Ca2+ uptake over a concentration range similar to that which blocks cellular elongation (IC50 = 7.5 ± 0.4 ,uM) (11). The lack of CAI effect on IP3 formation but sensitivity of calcium influx to CAI supports the IP3 independence of muscarinic receptor-induced calcium influx in CHO-Kl cells. mS Receptor-Stimulated Tyrosine Phosphorylation Is Calcium-Dependent. Carbachol-stimulated CHOm5 cells demonstrated a receptor-mediated increase in tyrosine phosphorylation of several proteins that was attenuated by the addition of atropine (Fig. 6A). Tyrosine phosphorylation was also stimulated by the calcium ionophore ionomycin in the absence of ligand (Fig. 6B). Removal of extracellular calcium resulted in the loss of carbachol and ionomycin-stimulated tyrosine phosphorylation, indicating the requirement for calcium influx. mS-stimulated protein tyrosine phosphorylation occurred at a concentration that stimulated calcium influx (10

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FIG. 3. Reduction in mass and delayed onset of tumors in CHOm5-inoculated nude mice. Nude mice were inoculated subcutaneously with CHOm5 cells (106 cells per site). Inoculation sites were treated with 20 Al of dimethyl sulfoxide, vehicle (control), carbachol (1 mM), or carbachol plus atropine (1 mM) twice daily for the duration of the study.

FIG. 4. Comparison of mS receptor-stimulated signaling pathways with morphology change in CHOmS cells. CHOm5 cells were stimulated with various concentrations of the muscannic receptor agonist carbachol, and 45Ca2+ uptake, [3H]arachidonic acid (AA) release, (3H]6P3 and release, [3H]phosphatidylethanol (PE) release, and cAMP accumulation were measured as an index of receptoroperated calcium channels, phospholipase A2, phospholipase C, phospholipase D, and adenylate cyclase activity, respectively. Carbachol-stimulated morphology change was measured as change in CHOmS cell length/width (L/W) ratio.

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M:Proc. Natl. Acad. Sci. USA 90 (1993)

Table 1. EC50 values for carbachol-stimulated second messengers in CHOm5 cells

potential but did cause specific suppression of the malignant phenotype in soft agar and in vivo. Both the morphology change and the tumor suppression were agonist-dependent, reversible, and blocked by a specific antagonist, and therefore were receptor-dependent. The carbachol-stimulated CHOm5 cell reverted to a nonmalignant phenotype as demonstrated by inhibition of colonization in soft agar and by the delayed onset and reduced size and number of tumors formed in nude mice. The muscarinic receptor-mediated morphology change is therefore a visual index of the phenotypic regression from a tumorigenic to nontumorigenic CHO-Kl cell. The carbacholinduced morphology change provided the opportunity to investigate which of the muscarinic receptor-stimulated signal transduction pathways mediated this process. When expressed in the CHO-Kl cell, the ml, m3, and m5 receptors activate multiple signal transduction pathways, including phospholipases A2, C, and D, adenylate cyclase, IP3-independent receptor-operated calcium channels, and calcium-dependent tyrosine kinase(s). Native muscarinic receptor-mediated activation of these same signaling pathways has been reported previously in a variety of cells, ruling out the possibility of indiscriminant coupling (19-22). Stimulation of muscarinic m2 and m4 receptors, which results in an inhibition of adenylate cyclase, had no effect on CHO-Kl cell morphology, indicating a muscarinic receptor subtype specificity for tumor suppression, and provides further evidence for independence from adenylate cyclase. Pertussis toxin, which ADP-ribosylates G1 proteins, had no effect on any of the signaling pathways associated with the ml, m3, and m5 receptors, suggesting independence from regulation by this type of G protein. Calcium influx was at least 2 orders of magnitude more sensitive to carbachol stimulation when compared to the other signal transduction pathways linked to muscarinic receptor stimulation. Its importance is further emphasized by the extracellular calcium requirement of phospholipase A2 (10), phospholipase D (data not shown), and tyrosine kinase and independence of phospholipase C (Fig. 6A) id the CHO-Ki cell. Calcium influx and the morphology change were stimulated at a concentration of carbachol that had no effect on the other signaling pathways tested, ruling out their possible involvement. CHO cells lack voltage-sensitive calcium channels, and muscarinic receptoractivated calcium influx is independent of IP3 release (12). These findings support a role for receptor-operated calcium channels (23) in primary growth regulation (24). Tyrosine phosphorylation observed following muscarinic receptor activation is reminiscent of signaling more commonly associated with growth factor receptors (25). Muscarinic receptor (22) and the bombesin, vasopressin, and endothelin neuropeptide receptors (26), all which are G proteincoupled receptors, have been shown to increase tyrosine phosphorylation of multiple proteins. The physiologic significance of neurotransmitter receptor-regulated tyrosine phosphorylation is unknown. In our studies, tyrosine phosphorylation was dependent on extracellular calcium, and the tyrosine kinase inhibitor BABA, a precursor in the chemical synthesis of the tyrosine kinase inhibitor lavendustin A (27), attenuated the receptor-stimulated morphologic change of CHOm5 cells. These data suggest that receptor-operated calcium influx may play a role in growth regulation through activation of tyrosine kinases. BABA acts by competing at the ATP binding site on tyrosine kinases and therefore may have other mechanism and sites of action. Tyrophostin 47 and RCAM lysozyme, tyrosine kinase inhibitors that do not bind to the ATP binding site (18, 28), were partially active, suggesting some specific action at tyrosine kinases. It is not known which tyrosine kinase is under the control of m5

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nM) but did not stimulate any of the other second-messenger pathways tested (data not shown). Several tyrosine kinase inhibitors were tested for their ability to attenuate carbacholstimulated tyrosine phosphorylation. Lavendustin A and methyl 2,5-dihydroxycinnamate had no effect up to concentrations of 100 ,uM (data not shown). In contrast, BABA was effective in reducing carbachol-stimulated tyrosine phosphorylation at 10 ,uM (Fig. 6C). Reduced, carbamoylmethylated (RCAM) lysozyme; genistein; and tyrophostin 47 (18) were much less potent as inhibitors of carbachol-stimulated tyrosine phosphorylation, requiring at least 50 ,uM for any major decrease to be observed (data not shown). When the tyrosine kinase inhibitors were tested for their effect on carbachol-stimulated morphology change, only BABA was effective in attenuating (75% at 1 ,uM) the carbachol-activated response. RCAM lysozyme, genistein, and tyrophostin 47 also demonstrated partial (