Discordant Signal Transduction and Growth Inhibition of Small Cell Lung Carcinomas Induced by Expression of GTPase-deficient Gα16

Abstract

Small cell lung carcinoma (SCLC) accounts for 20 -25% of primary lung cancers and is rapidly growing, widely metastatic, and rarely curable. Autocrine stimulation of multiple G protein-coupled neuropeptide receptor systems contributes to the transformed growth of SCLC. The ability of neuropeptide receptors to stimulate phospholipase C and mobilize intracellular Ca 2؉ indicates that G q family members of heterotrimeric G proteins are a convergence point mediating autocrine signaling by multiple neuropeptides in SCLC. Expression of a GTPase-deficient, constitutive active form of an ␣ q family member, ␣ 16 Q212L, in SCLC markedly inhibited growth of the cells in soft agar and tumor formation in nude mice. SCLC lines expressing ␣ 16 Q212L exhibited 2-4-fold elevated basal phospholipase C activity, but neuropeptide and hormone-regulated intracellular Ca 2؉ mobilization was nearly abolished. The data suggest that Ca 2؉ mobilization is an obligatory signal in neuropeptide-stimulated growth of SCLC. In addition, the proline-directed c-Jun NH 2 -terminal kinases/stress-activated protein kinases, which are members of the mitogen-activated protein kinase family, were stimulated ϳ2-fold in parental SCLC in response to exogenous neuropeptides and muscarinic agonists and were constitutively activated to the same degree in ␣ 16 Q212L-expressing SCLC. Thus, ␣ 16 Q212L expression induced desensitization of neuropeptide-stimulated Ca 2؉ signaling and persistent activation of the c-Jun NH 2 -terminal kinase/ stress-activated protein kinase pathway. We propose that the induction of discordant signaling by selective perturbation of receptor-regulated effector systems leads to the inhibition of SCLC cell growth. Small cell lung carcinoma (SCLC)1 displays neuroendocrine features exemplified by the presence of cytoplasmic neurosecretory granules containing a wide variety of mitogenic neuropeptides including gastrin-releasing peptide, arginine vasopressin, neurotensin, cholecystokinin, and many others (1, 2). Significantly, SCLC also expresses receptors for these neuropeptides, thereby establishing autocrine-stimulated cell growth (3). The number and variability of neuropeptides released from individual small cell carcinomas hampers effective blockade of mitogenic signaling at the level of ligand/receptor binding using specific neuropeptide antagonists. This redundancy at the level of receptor signaling highlights the importance of defining the intracellular components involved in mitogenic signal transduction in SCLC where convergence of multiple neuropeptide receptor systems into common pathways would be anticipated.Molecular cloning of the receptors for gastrin-releasing peptide, vasopressin, and gastrin/cholecystokinin reveals that they are members of the superfamily of seven membrane-spanning receptors (4 -8). As a class, these receptors initiate signaling in response to ligand binding by interacting with heterotrimeric G proteins. Although the repertoire of G proteins potentially involved in neuropeptide signaling in SCLC is quite...

Autocrine and paracrine signaling through neuropeptide receptors in human cancer

“…According to this hypothesis, substance P derivatives preferentially bind to and stabilize neuropeptide receptors in a conformational state that activates G 12 and G 13 proteins, but not G q proteins (Jarpe et al, 1998). Thus, the substance P derivative o er a therapeutic approach to induce unbalanced, or discordant, signaling that is similar to what was previously accomplished in SCLC cells by retrovirus-mediated expression of constitutively active Ga q proteins or mutant PLCb proteins (Beekman et al, 1998;Heasley et al, 1996a).…”

“…This paradoxical inhibition of Ca 2+ mobilization by constitutively activated Ga q proteins appears to arise by way of a compensatory down-regulation of IP 3 -liganded Ca 2+ channels in the endoplasmic reticulum (Lobaugh et al, 1996;Quick et al, 1996). The resulting discordant signaling relative to the balanced signaling initiated by neuropeptide receptors is hypothesized to result in the marked reduction in SCLC cell growth that was observed (Heasley et al, 1996a). In another study, expression of a catalytically-inactive form of PLCb in SCLC cells which retains the sequences that interact with Ga q signi®cantly inhibited basal PKC activity and receptor-stimulated phosphatidylinositol hydrolysis, Ca 2+ mobilization and ERK activation, but not JNK activation (Beekman et al, 1998).…”

“…Expression of GTPasede®cient forms of Ga q proteins in SCLC lines induces desensitization of neuropeptide-stimulated Ca 2+ mobilization and increased basal JNK activity (Heasley et al, 1996a). This paradoxical inhibition of Ca 2+ mobilization by constitutively activated Ga q proteins appears to arise by way of a compensatory down-regulation of IP 3 -liganded Ca 2+ channels in the endoplasmic reticulum (Lobaugh et al, 1996;Quick et al, 1996).…”

The galanin receptor type 2 initiates multiple signaling pathways in small cell lung cancer cells by coupling to Gq, Gi and G12 proteins

“…The neuropeptide-controlled growth of SCLC cells appears to be controlled by a delicately orchestrated interplay between various signaling pathways initiated by Gprotein-coupled neuropeptide receptors. Overexpression of GTPase-de®cient G-protein a subunits of the G q/11 family, for instance, results in discordant signal transduction and growth inhibition (Heasley et al, 1996). Likewise, expression of a constitutively active Raf kinase in SCLC cells leads to persistent ERK activation but inhibition of clonogenicity and growth arrest, most probably due to a marked induction of the cyclin-dependent kinase inhibitor p27 kip1 (Ravi et al, 1998).…”

Potentiation of apoptosis by low dose stress stimuli in cells expressing activated MEK kinase 1

“…An emerging theme is that the pathways signaling apoptosis overlap with those that mediate growth and di erentiation (LassignalJohnson et al, 1996;Fanidi et al, 1992;Gardner et al, 1993;Evan et al, 1992). In the context of signal transduction pathways, integration of signals initiated by growth factor and cytokine receptors, therefore, can commit a cell to either proliferation or apoptosis (Xia et al, 1995;Heasley et al, 1996;Gardner and Johnson, 1996). For example, we recently demonstrated that FGF-2 protects L929 cells from TNFa-stimulated apoptosis .…”