(2001) 20, 1563 ± 1569 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc

Autocrine and through receptors in human

Lynn E Heasley*,1

1Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado, CO 80262, USA

Autocrine and paracrine signaling leading to stimulation lators in the nervous system. In the large majority of of tumor growth is a common theme in human cases, the receptors which mediate signaling by . In addition to polypeptide growth factors such are members of the superfamily of G as EGF family members which signal through protein-coupled, seven membrane-spanning receptors , accumulating evidence supports the (Burbach and Meijer, 1992). In this review, speci®c autocrine and paracrine involvement of speci®c neuro- examples where physiologic functions of selected peptides with de®ned physiologic actions as neurotrans- neuropeptide families are subverted to roles in mitters and gut in , gastric, colorectal, tumorigenesis and hyperplastic growth are explored. pancreatic and prostatic cancers. These neuropeptides, The precise neuropeptide receptor subtypes and the including -releasing peptide, , neu- dominant intracellular signaling pathways that mediate rotensin, gastrin, and arginine vasopres- the various proliferative responses are reviewed and the sin bind seven transmembrane-spanning receptors that emerging utilities of both speci®c and broad-spectrum couple to heterotrimeric G proteins. Studies with human neuropeptide antagonists as therapeutics for human small cell lung cancer (SCLC) cells support a require- cancers are considered. ment for balanced signaling through Gq and G12/13 proteins leading to intracellular Ca2+ mobilization, PKC Neuropeptides with putative autocrine or paracrine activation and regulation of the ERK and JNK MAP involvement in human cancers pathways. While speci®c neuropeptide antagonists o€er promise for interrupting the single neuropeptide While a large number of peptide hormones have been autocrine systems operating in pancreatic and prostatic identi®ed with putative roles as growth factors in cancers, SCLC is exempli®ed by multiple, redundant human cancer, this review will restrict its focus to the neuropeptide autocrine systems such that tumor growth evidence supporting the autocrine and paracrine cannot be inhibited with a single speci®c antagonist. involvement of the -related peptides, gastrin, However, a novel class of neuropeptide derivatives based cholecystokinin, and arginine vasopressin on the substance P sequence have been de®ned that in human cancer. Physiologically, each of these exhibit broad speci®city for neuropeptide receptors and peptides ful®lls dual functions; as a induce in SCLC by functioning as biased or neuromodulator in the nervous system and as a agonists that stimulate discordant . or endocrine factor in the periphery. A brief Thus, interruption of autocrine and paracrine neuropep- overview of the normal functions of the di€erent tide signaling with speci®c antagonists or broad-spectrum peptides and their receptors is presented below as well biased agonists o€er promising new therapeutic ap- as the evidence for their autocrine and paracrine proaches to the treatment of human cancers. Oncogene involvement in speci®c human cancers. (2001) 20, 1563 ± 1569. Bombesin-like peptides The prototypical neuropep- Keywords: neuropeptides; autocrine; paracrine; cancer tides in this family, bombesin and ranatensin, were cells; G proteins; receptors ®rst isolated from amphibian and led to the subsequent identi®cation of the mammalian homolo- gues, gastrin-releasing peptide (GRP) and neuromedin Introduction B (NMB), respectively (reviewed in Battey and Wada, 1991; Ohki-Hamazaki, 2000). In the periphery, GRP Neuropeptides function peripherally as paracrine and and NMB control a wide spectrum of actions including endocrine factors to regulate diverse physiologic smooth muscle contraction and exocrine and endocrine processes and act as or neuromodu- secretion. GRP is, in fact, named for its ability to stimulate gastrin release from gastrin (G) cells in the antral mucosa. Acting at the central nervous system, these peptides regulate food intake, body temperature *Correspondence: LE Heasley, Division of Renal Medicine, C-281, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., and glucose levels as well as certain behavioral Denver, CO 80262, USA responses. Seven membrane-spanning receptors exhibit- Neuropeptide signaling in cancer LE Heasley 1564 ing selectivity for GRP or NMB have been identi®ed assessed (Sun et al., 2000b). However, the ability of through molecular cloning approaches (reviewed in GRP antagonists to inhibit the growth of an ovarian Battey and Wada, 1991; Ohki-Hamazaki, 2000). In carcinoma cell line (Chatzistamou et al., 2000) suggests addition, a distinct receptor referred to as bombesin that a GRP autocrine system may also function in receptor subtype-3 (BRS-3) with extensive homology to ovarian carcinomas. Finally, both central and periph- the GRP and NMB receptors has been cloned, for eral primitive neuroectodermal tumors (PNET) have which the endogenous remains unde®ned (Fathi been shown to express GRP (Fruhawald et al., 1998; et al., 1993; Gorbulev et al., 1992). Interestingly, mice Lawlor et al., 1998) and the in vitro growth of a in which the BRS-3 gene has been disrupted develop peripheral PNET cell line was inhibited by a GRP multiple metabolic defects and mild obesity, indicating receptor antagonist (Lawlor et al., 1998). that BRS-3 and its unde®ned ligand also regulate important endocrine and metabolic processes (Ohki- Gastrin and cholecystokinin Gastrin and cholecystoki- Hamazaki et al., 1997). nin (CCK) serve physiological functions as hormones The bombesin-like peptides, GRP and NMB, were in the gastrointestinal tract and as neuropeptides in the among the ®rst neuropeptides to be implicated as nervous system (reviewed in Wank, 1995, 1998). autocrine growth factors in lung cancer cells. In small Gastrin and CCK are derived from distinct prepro- cell lung cancer (SCLC) cells, GRP (Cuttitta et al., hormones, but are structurally similar peptides that 1985; Moody et al., 1981) and NMB (Cardona et al., share the same C-terminal ®ve amino acids, are a- 1991) are widely synthesized and secreted into the amidated at their C-termini and are partially sulfated medium. In addition, the majority of SCLC cells on tyrosine. CCK is synthesized in neurons throughout express the mRNA for one or more of the bombesin- the central and peripheral nervous system and in like receptors (Corjay et al., 1991; Fathi et al., 1993; intestinal neuroendocrine cells (I cells) while gastrin Toi-Scott et al., 1996). The functionality of the GRP peptides are expressed and released from the duode- and NMB receptors expressed on SCLC cells as num and gastric antrum (gastrin cells) as well as the measured by intracellular Ca2+ mobilization in re- pituitary. Two distinct receptors for gastrin and CCK sponse to applied GRP or NMB has also been have been identi®ed to date; the CCK-A receptor is established (Bunn et al., 1992; Corjay et al., 1991; highly selective for sulfated CCK, while the CCK-B Moody et al., 1992; Woll and Rozengurt, 1989). The receptor exhibits a similar high anity for both ability of bombesin-like peptide antagonists (Mahmoud sulfated and nonsulfated CCK and gastrin (Wank, et al., 1991) or neutralizing antibodies (Cuttitta et al., 1995, 1998). CCK-A receptors are expressed on 1985) to reduce the growth of some cultured SCLC cell multiple cell types in the pancreas, stomach and lines provides evidence for bombesin-like peptides as intestine, but are also expressed in select areas of the autocrine growth factors in SCLC. However, as will central and peripheral nervous system. By contrast, the become clear in this review, SCLC is an example of a CCK-B receptors are predominantly expressed in the cancer where multiple, variably-expressed neuropeptide central nervous system in a pattern similar to CCK and autocrine systems operate in a redundant fashion and on parietal cells and enterochroman-like (ECL) cells their simultaneous disruption will most likely be in the gastric mucosa. required for e€ective inhibition of . In addition to regulating Normal prostate tissue and a substantial proportion secretion, evidence indicates that gastrin and CCK (*70%) of primary and metastatic prostatic tumors exert mitogenic actions on speci®c cells in the gastric contain a dispersed subpopulation of neuroendocrine mucosa and pancreas, respectively. Hypergastrinemia cells that appear to expand in prostate cancers as they resulting from pharmacological blockade of acid become -independent (Aprikian et al., 1998; secretion from parietal cells leads to a modest, but Nelson and Carducci, 2000). These neuroendocrine stable increase in gastrin cell density. The ECL cells in cells express a variety of bioactive neuropeptides the gastric mucosa also respond mitogenically to including GRP. A number of studies document the elevated gastrin levels except that their density steadily elevated expression of GRP receptors in prostate increases during hypergastrinemia and eventually may cancer specimens (Bartholdi et al., 1998; Markwalder result in the formation of ECL cell carcinoids (Dock- and Reubi, 1999; Sun et al., 2000a) and on cultured ray, 1999; Sundler et al., 1991). CCK via the CCK-A prostatic cancer cell lines (Aprikian et al., 1996), receptor mediates the pancreatic hyperplasia observed supporting a paracrine role for GRP in prostate cancer following the feeding of protease inhibitors or progression. pancreaticobiliary diversion and CCK enhances the While SCLC and prostate cancer are the best induction by carcinogens of acinar tumors in the characterized human tumors where bombesin-like pancreas (reviewed in Watanapa and Williamson, peptides are likely to function as autocrine or paracrine 1993). growth factors, the literature indicates the co-expres- The potential role of gastrin and CCK as autocrine sion of GRP and GRP receptors in colorectal samples or paracrine factors in gastrointestinal cancers is (Chave et al., 2000). Also, expression of all three controversial (reviewed in Baldwin, 1995, Baldwin bombesin-like peptide receptor subtypes has been and Shulke, 1998). While the evidence indicates that noted in ovarian epithelial cancers although the most gastrointestinal carcinomas express and synthe- expression of their neuropeptide ligands were not size progastrin, con¯icting reports on the degree of

Oncogene Neuropeptide signaling in cancer LE Heasley 1565 gastrin amidation which is required for binding to the restricted to the renal medulla where it mediates the de®ned CCK-B receptor in cancer specimens and cell antidiuretic e€ect of AVP. The V3 receptor was initially lines prevent a clear assignment of gastrin as an described in pituitary corticotroph cells where it autocrine factor in gastrointestinal cancer. However, potentiates the release of ACTH, but is expressed in novel gastrin receptors unrelated to either CCK-A or many other cell types with functions that remain to be CCK-B receptors that bind non-amidated gastrin de®ned. peptides have been described and should be considered Similar to the bombesin-like peptides, a considerable as potential mediators of autocrine and paracrine percentage of cultured SCLC cells express (Gross et al., growth actions of gastrin peptides in gastrointestinal 1993; Sausville et al., 1985; Verbeeck et al., 1992) and cancers (Baldwin and Shulke, 1998; Dockray, 1999). secrete (Gross et al., 1993) AVP. Consistent with the in By contrast, convincing evidence supports the involve- vitro production of AVP by SCLC cells, SCLC patients ment of a gastrin and CCK-B receptor autocrine frequently present with elevated plasma AVP (Gross et system in the pathogenesis of human pancreatic al., 1993; North, 1991) which, via the renal V2 carcinoma. Amidated gastrin was detected in the receptor, leads to the syndrome of inappropriate majority of pancreatic adenocarcinomas, but not in secretion of antidiuretic hormone (SIADH), associated normal pancreatic tissue and the CCK-B receptor with plasma hyponatremia and hypoosmolality and mRNA was expressed in all carcinomas. While the urinary hyperosmolality (Gross et al., 1993; Hamilton CCK-A receptor mRNA was also widely expressed in et al., 1972). As with the bombesin-like peptides and most of the tumor samples, neither amidated and gastrin/CCK, AVP and the de®ned AVP receptors are sulfated CCK nor CCK precursors were expressed in frequently co-expressed in SCLC (North et al., 1998), the carcinomas or normal pancreatic tissues (Goetze et thereby establishing the potential for autocrine-stimu- al., 2000; Weinberg et al., 1997). Moreover, co- lated growth. These observations coupled with ®ndings expression of gastrin and CCK-B receptors is observed that many SCLC lines respond to exogenously-added in multiple cultured pancreatic carcinoma cell lines and AVP with intracellular Ca2+ mobilization (Bunn et al., growth of these cell lines is inhibited by CCK-B 1992; Hong and Moody, 1991; Woll and Rozengurt, receptor antagonists and gastrin neutralizing anti- 1989) and increased cloning eciency (Sethi and bodies (Blackmore and Hirst, 1992; Smith et al., Rozengurt, 1991) suggest that AVP, like GRP, gastrin 1994, 1996). and CCK, may contribute to autocrine growth of Evidence also exists to support the autocrine action SCLC. of CCK and gastrin in SCLC. The CCK gene is expressed in cultured SCLC cells and the preproCCK Neurotensin The neuropeptide, neurotensin (NT), is processed into bioactive, carboxyamidated CCK provides yet another example of a peptide that serves peptides (Geijer et al., 1990). Gastrin mRNA was also dual functions as a neurotransmitter or neuromodu- frequently detected in SCLC (Reubi et al., 1997). lator in the nervous system and a hormone in the Moreover, both CCK-A (Sethi, 1993) and CCK-B periphery. Centrally, NT is a modulator of receptors have been measured on SCLC cells (Reubi et transmission and pituitary hormone secretion and al., 1997; Staley et al., 1990) and gastrin and CCK exerts hypothermic and analgesic e€ects in the brain stimulate intracellular Ca2+ mobilization (Sethi and while it functions as a peripheral hormone in the Rozengurt, 1992; Sethi et al., 1993; Staley et al., 1989). digestive tract and cardiovascular system. Cloning As with GRP, the redundancy of neuropeptide e€orts have de®ned two distinct receptors with seven autocrine systems in SCLC complicates the clear membrane-spanning topography that are expressed in assignment of any particular neuropeptide to a speci®c brain regions and the intestine as well as a dominant role in cellular transformation in SCLC. novel receptor identical to sortilin (Vincent et al., 1999). Evidence indicates that NT also serves as an Arginine vasopressin The neurohypophysial hormone autocrine in prostate cancer. NT arginine vasopressin (AVP) is a peripheral regulator of receptors were detected in an androgen-independent water excretion in the kidney, a potent vasoconstrictor prostatic carcinoma cell line and NT stimulated the and a neurotransmitter or neuromodulator in the growth of these cells, indicating that NT could central nervous system. The AVP peptide is derived contribute to the growth or survival of prostate tumor from a large precursor encoding a signal peptide, the cells (Seethalakshmi et al., 1997). In a separate study, nine amino acid peptide hormone, a neurophysin withdrawal of androgen from an androgen-dependent polypeptide that serves as a hormone carrier and a prostate cancer cell line increased the production and C-terminal of unknown function (Ivell et secretion of NT and the cell became mitogenically- al., 1983). Three distinct AVP receptors (V1,V2 and responsive to NT (Sehgal et al., 1994). Finally, NT is V3) have been identi®ed through molecular cloning produced and secreted by SCLC cells (Davis et al., techniques (reviewed in Thibonnier et al., 1998). The 1991; Moody et al., 1985) and SCLC cells respond to V1 receptor is expressed in vascular smooth muscle exogenously-added NT (Bunn et al., 1992; Sethi and cells and mediates the vasoconstrictor actions of AVP Rozengurt, 1991; Seu€erlein and Rozengurt, 1996; (Nemeno€, 1998), but is also expressed in many other Woll and Rozengurt, 1989), indicating that this tissues and is the major AVP receptor in the brain. By neuropeptide may contribute along with the other contrast, expression of the V2 receptor is largely neuropeptide families to the growth of SCLC.

Oncogene Neuropeptide signaling in cancer LE Heasley 1566 Thus, evidence supports the involvement of diverse general ability to stimulate intracellular Ca2+ mobiliza- neuropeptide families as growth factors in human tion (Bunn et al., 1992; Woll and Rozengurt, 1989). cancers. In the case of prostatic and pancreatic Increased intracellular Ca2+ concentrations are carcinomas, the candidate neuropeptides, GRP and mediated by receptor interaction with Gq proteins gastrin, exert physiological roles in the tissues from and the subsequent activation of Cb which the cancers arise, indicating that the normal (PLCb), which generates inositol trisphosphate (IP3), a function of the neuropeptides has been subverted to stimulatory ligand for Ca2+ actions in cellular transformation. The molecular channels (Figure 1 and Dhanasekaran et al., 1995). alterations that may allow these peptides to function The targets of increased cellular Ca2+ are poorly as growth factors in the cancer cells that arise from the de®ned in SCLC cells, but are likely to include Ca2+/ respective tissues are not completely clear, although calmodulin-dependent protein kinases. Increased cellu- increased density of bombesin binding sites and GRP lar Ca2+ also integrates with diacylglycerol (DAG) mRNA expression have been noted in prostatic generated by PLCb activity to stimulate protein kinase carcinomas (Bartholdi et al., 1998; Markwalder and C (PKC) which, in many cells including SCLC cells Reubi, 1999). While CCK-B receptors are expressed in (Seu€erlein and Rozengurt, 1996), serves as a prox- both normal pancreas and in pancreatic carcinomas, imal, neuropeptide-stimulated regulator of the protein the majority of pancreatic carcinomas synthesized kinase cascade leading to activation of the extracellular amidated gastrin but the normal pancreatic tissue signal-regulated kinase (ERK) members of the mito- expressed only trace amounts of poorly processed gen-activated protein (MAP) kinase family (see Figure progastrin (Goetze et al., 2000). Thus, a predicted 1 and Schonwasser et al., 1998). PKC has also been result of increased receptor density in prostatic shown to mediate the activation of protein kinase D carcinomas or increased ligand production in pancrea- (PKD)/PKCm by neuropeptides in Swiss 3T3 cells and tic cancers would be an enhanced magnitude and/or SCLC cells (Paolucci and Rozengurt, 1999; Zugaza et duration of signaling in the evolving cancer cell. In a al., 1997). The putative targets of PKD/PKCm in similar manner, ECL carcinoids in the stomach arise when local levels of gastrin, which serves as a growth factor for ECL cells, are chronically increased (Dock- ray, 1999; Sundler et al., 1991). The pulmonary precursor of SCLC and the regulatory mechanisms that result in the induction of the extensive array of neuropeptides and their cognate receptors remain poorly understood. While the existence of pulmonary neuroendocrine cells that synthesize GRP has been documented in normal and diseased lung (Miller, 1989), these cells are generally not considered to be the cell of origin for SCLC. A more likely possibility is that induction of a regulatory mechanism(s) resulting in ectopic expres- sion of a large number of neuroendocrine genes occurs during the selective steps that eventually result in small cell lung cancers. Several laboratories have employed the 5'-¯anking promoter regions of the AVP, GRP and the pro-opiomelanocortin genes to begin to de®ne the regulatory mechanisms that may mediate the general induction of neuroendocrine gene expression in SCLC (Coulson et al., 1999a,b; Markowitz et al., 1988; Nagalla and Spindel, 1994; Picon et al., 1995). The ®ndings suggest that regulation of these genes will be complex and involve the modulation of and enhancers as well as tissue-speci®c factors. Figure 1 Signal pathways regulated by neuropeptide receptors. A prototypical neuropeptide receptor is depicted that couples to the indicated signaling pathways through G and G proteins. Signal pathways mediating neuropeptide-stimulated q 12,13 Abbreviations: PLCb, phospholipase Cb; PIP2, phosphatidylino- cellular transformation sitol-bisphosphate; IP3, inositol trisphosphate; DAG, diacylgly- cerol; PKC, protein kinase C; PKD, protein kinase D/PKCm; Molecular cloning of receptors for the di€erent ERK, extracellular signal-regulated kinase; MEK, - neuropeptides described above reveals their member- activated protein-ERK kinase; MEKK, MEK kinase; MKK, ship in the superfamily of heterotrimeric - mitogen-activated protein kinase kinase; JNK, cJun N-terminal coupled, seven transmembrane-spanning receptors. kinase; GEF, guanine nucleotide exchange factor. The positioning of the cytosolic tyrosine kinases, Src and Tec/Bmx, as direct Moreover, analysis of signaling by the many neuropep- e€ectors of G12 and G13 proteins that mediate Rho activation of tide receptors expressed in SCLC cells highlights their the JNK pathway and the is not intended

Oncogene Neuropeptide signaling in cancer LE Heasley 1567 neuropeptide-responsive cells and their role in cell Substance P derivatives as novel therapeutics growth and transformation remain to be de®ned. In that stimulate apoptotic signaling through addition to Gq proteins, biochemical evidence indicates neuropeptide receptors that neuropeptide receptors interact with G12 and G13 proteins (Barr et al., 1997; Jian et al., 1999; Wittau et The identi®cation and characterization of substituted al., 2000), leading to the activation of the Ras and Rho derivatives of the neuropeptide, substance P, that family of monomeric G proteins (Collins et al., 1996; exhibited broad antagonist activity for a variety of Wadsworth et al., 1997) and regulation of the cJun N- neuropeptide receptors expressed on SCLC cells terminal kinase (JNK) members of the MAP kinase provide a novel approach to the disruption of multiple family (Heasley et al., 1996a,b; Higashita et al., 1997; neuropeptide autocrine systems (Sethi et al., 1992). Vara Prasad et al., 1995) as well as the reorganization Rozengurt and colleagues (Seckl et al., 1997; Woll and of the actin cytoskeleton (Buhl et al., 1995; Gohla et Rozengurt, 1988, 1990) have shown that speci®c al., 1999). Identi®cation of the e€ectors that mediate substance P derivatives inhibit receptor-mediated Ca2+ these actions of G12 and G13 is an active area of mobilization by multiple neuropeptides including AVP, investigation, and studies indicate roles for Rho family CCK, GRP and NT and that the derivatives markedly guanine nucleotide exchange factors (Kozasa et al., reduce the in vitro growth of SCLC cells. Signi®cantly, 1998) and Src and Tec/Bmx-family of tyrosine kinases the substance P derivatives also inhibit SCLC growth (Mao et al., 1998; Nagao et al., 1999) upstream of JNK as xenografts in nude mice with little or no apparent and cytoskeletal regulation. As depicted in Figure 1, toxicity (Everard et al., 1993; Langdon et al., 1992; the Ca2+/calmodulin-dependent protein kinases, ERKs Seckl et al., 1997). Of importance are the ®ndings that and JNKs are predicted to converge on the substance P derivatives induce apoptosis in lung factors that control gene expression related to cancer cell lines (Reeve and Bleehen, 1994; Rosati et mitogenesis and cellular transformation. Thus, neuro- al., 1998) and di€erentially-inhibit receptor-mediated 2+ peptide receptors couple to Gq,G12 and G13 proteins signaling of Ca mobilization and ERK activation which yield balanced signaling of Ca2+ mobilization (Mitchell et al., 1995) suggesting that the substance P and ERK and JNK activation (Figure 1). derivatives may not function simply as classical Accumulating evidence suggests that Gq proteins receptor-blocking antagonists. In fact, a recent study and PLCb exert dominant e€ects in signal transduc- with the substance P derivative, [D-Arg1, D-Phe5,D- tion of SCLC cell growth. Expression of GTPase- Trp7,9, Leu11] substance P, demonstrated that concen- 2+ de®cient forms of Gaq proteins in SCLC lines trations of peptide which block GRP-induced Ca induces desensitization of neuropeptide-stimulated mobilization cause activation of the JNK pathway and Ca2+ mobilization and increased basal JNK activity induce Rho-dependent cytoskeletal changes in Swiss (Heasley et al., 1996a). This paradoxical inhibition 3T3 cells (Jarpe et al., 1998). Induction of JNK activity 2+ of Ca mobilization by constitutively activated Gaq by a di€erent substance P derivative was also shown in proteins appears to arise by way of a compensatory SCLC cells (MacKinnon et al., 1999). The ability of 2+ down-regulation of IP3-liganded Ca channels in the the substance P derivative to activate the JNK pathway endoplasmic reticulum (Lobaugh et al., 1996; Quick was dependent upon the expression of a neuropeptide et al., 1996). The resulting discordant signaling receptor (Jarpe et al., 1998), revealing that the actions relative to the balanced signaling initiated by of these novel peptides are receptor-mediated. Based neuropeptide receptors is hypothesized to result in on their ability to selectively stimulate JNK activity the marked reduction in SCLC cell growth that was and cytoskeletal changes, possibly through G12 and 2+ observed (Heasley et al., 1996a). In another study, G13, but not Ca mobilization through Gq proteins, expression of a catalytically-inactive form of PLCb substance P derivatives have been hypothesized to in SCLC cells which retains the sequences that represent a novel class of agonist referred to as biased interact with Gaq signi®cantly inhibited basal PKC agonists (Jarpe et al., 1998). According to this activity and receptor-stimulated phosphatidylinositol hypothesis, substance P derivatives preferentially bind hydrolysis, Ca2+ mobilization and ERK activation, to and stabilize neuropeptide receptors in a conforma- but not JNK activation (Beekman et al., 1998). tional state that activates G12 and G13 proteins, but not Importantly, anchorage-independent cell growth was Gq proteins (Jarpe et al., 1998). Thus, the substance P markedly blunted. These ®ndings together with derivative o€er a therapeutic approach to induce studies showing strong SCLC cell growth reduction unbalanced, or discordant, signaling that is similar to by inhibitors of PLCb (Strassheim et al., 2000), what was previously accomplished in SCLC cells by PKC (Seu€erlein and Rozengurt, 1996) and MEK retrovirus-mediated expression of constitutively active (Seu€erlein and Rozengurt, 1996) support the Gaq proteins or mutant PLCb proteins (Beekman et requirement for balanced signaling through Gq al., 1998; Heasley et al., 1996a). proteins, PLCb, PKC, ERKs and Ca2+ mobilization In conclusion, the substance P derivatives o€er in neuropeptide-stimulated growth of SCLC cells. exciting promise as novel therapeutics for the recep- However, the requirement for G12 and G13-regulated tor-mediated induction of apoptosis in cancer cells pathways including JNK activation and cytoskeletal where neuropeptide autocrine and paracrine systems regulation in neuropeptide-stimulated growth remain are involved. A search for shorter substance P largely untested. derivatives or analogs with increased receptor anity

Oncogene Neuropeptide signaling in cancer LE Heasley 1568 or serum stability seems warranted and is, in fact, an treatment of human cancers involving neuropeptide ongoing focus of several laboratories (MacKinnon et receptor systems. al., 1999; Nyeki et al., 1998; Rosati et al., 1998; Woll and Rozengurt, 1990). Ideally, a small nonpeptide molecule based on the structure of the substance P Acknowledgments derivative that functions similarly, but avoids the I appreciate many helpful discussions regarding this manu- problems associated with peptide synthesis and deliv- script with Raphael Nemeno€ (University of Colorado ery, may represent an excellent therapeutic alone or in Health Sciences Center) and support from NIH grants combination with existing chemotherapies for the DK19928 and CA58157.

References

Aprikian AG, Han K, Chevalier S, Bazinet M and Viallet J. Geijer T, Folkesson R, Rehfeld JF and Monstein H-J. (1996). J. Mol. Endocrinol., 16, 297 ± 306. (1990). FEBS Lett., 270, 30 ± 32. Aprikian AG, Han K, Guy L, Landry F, Begin LR and Goetze JP, Nielsen FC, Burcharth F and Rehfeld JF. (2000). Chevalier S. (1998). Prostate Suppl., 8, 52 ± 61. Cancer, 88, 2487 ± 2494. Baldwin GS. (1995). J. Gastroenterol. Hepatol., 10, 215 ± 232. Gohla A, O€ermanns S, Wilkie TM and Schultz G. (1999). J. Baldwin GS and Shulke A. (1998). Gut, 42, 581 ± 584. Biol. Chem., 274, 17901 ± 17907. Barr AJ, Brass LF and Manning DR. (1997). J. Biol. Chem., Gorbulev V, Akhundova A, Buchner H and Fahrenholz F. 272, 2223 ± 2229. (1992). Eur. J. Biochem., 208, 405 ± 410. BartholdiMF,WuJM,PuH,TroncosoP,EdenPAand Gross AJ, Steinberg SM, Reilly JG, Bliss Jr. DP, Brennan J, Feldman RI. (1998). Int. J. Cancer, 79, 82 ± 90. Le PT, Simmons A, Phelps R, Mulshine JL, Ihde DC and Battey J and Wada E. (1991). Trends Neurosci., 14, 524 ± 528. Johnson BE. (1993). Cancer Res., 53, 67 ± 74. Beekman A, Helfrich B, Bunn Jr. PA and Heasley LE. Hamilton BP, Upton GV and Amatruda Jr. TT. (1972). Clin. (1998). Cancer Res., 58, 910 ± 913. Endocrinol. Metab., 35, 764 ± 767. Blackmore M and Hirst BH. (1992). Br.J.Cancer,66, 32 ± Heasley LE, Zamarripa J, Storey B, Helfrich B, Mitchell 38. FM, Bunn Jr. PA and Johnson GL. (1996a). J. Biol. Buhl AM, Johnson NL, Dhanasekaran N and Johnson GL. Chem., 271, 349 ± 354. (1995). J. Biol. Chem., 270, 24631 ± 24634. Heasley LE, Storey B, Fanger GR, Zamarripa J and Maue Bunn Jr. PA, Chan D, Dienhart DG, Tolley R, Tagawa M RA. (1996b). Mol. Cell. Biol., 16, 648 ± 656. and Jewett PB. (1992). Cancer Res., 52, 24 ± 31. HigashitaR,LiL,VanPuttenV,YamamuraY,Zarinetchi Burbach JP and Meijer OC. (1992). Eur. J. Pharmacol., 227, F, Heasley L and Nemeno€ RA. (1997). J. Biol. Chem., 1±18. 272, 25845 ± 25850. Cardona C, Rabbitts PH, Spindel ER, Ghatei MA, Bleehen Hong M and Moody TW. (1991). Peptides, 12, 1315 ± 1319. NM, Bloom SR and Reeve JG. (1991). Cancer Res., 51, Ivell R, Schmale H and Richter D. (1983). Neuroendocrinol., 5205 ± 5211. 37, 235 ± 240. Chatzistamou I, Schally AV, Sun B, Armatis P and JarpeMB,KnallC,MitchellFM,BuhlAM,DuzicEand Szepeshazi K. (2000). Br. J. Cancer, 83, 906 ± 913. Johnson GL. (1998). J. Biol. Chem., 273, 3097 ± 3104. Chave HS, Gough AC, Palmer K, Preston SR and Primrose Jian X, Sainz E, Clark WA, Jensen RT, Battey JF and JN. (2000). Br.J.Cancer.,82, 124 ± 130. Northup JK. (1999). J. Biol. Chem., 274, 11573 ± 11581. Collins LR, Minden A, Karin M and Brown JH. (1996). J. Kozasa T, Jiang X, Hart MJ, Sternweis PM, Singer WD, Biol. Chem., 271, 17349 ± 17353. Gilman AG, Bollag G and Sternweis PC. (1998). Science, CorjayMH,DobrzanskiDJ,WayJM,VialleyJ,ShapiraH, 280, 2109 ± 2111. Worland P, Sausville EA and Battey JF. (1991). J. Biol. Langdon S, Sethi T, Ritchie A, Muir M, Smyth J and Chem., 266, 18771 ± 18779. Rozengurt E. (1992). Cancer Res., 52, 4554 ± 4557. Coulson JM, Fiskerstrand CE, Woll PJ and Quinn JP. Lawlor ER, Lim JF, Tao W, Poremba C, Chow CJ, (1999a). Cancer Res., 59, 5123 ± 5127. Kalousek IV, Kovar H, MacDonald TJ and Sorensen Coulson JM, Stanley J and Woll PJ. (1999b). Br.J.Cancer, PH. (1998). Cancer Res., 58, 2469 ± 2476. 80, 1935 ± 1944. Lobaugh LA, Eisfelder B, Gibson K, Johnson GL and Cuttitta F, Carney DN, Mulshine J, Moody TW, Fedorko J, Putney Jr. JW. (1996). Mol. Pharmacol., 50, 493 ± 500. Fischler A and Minna JD. (1985). Nature, 316, 823 ± 826. MacKinnon AC, Armstrong RA, Waters CM, Cummings J, Davis TP, Crowell S, McIntur€ B, Louis R and Gillespie T. Smyth JF, Haslett C and Sethi T. (1999). Br.J.Cancer,80, (1991). Peptides, 12, 17 ± 23. 1026 ± 1034. Dhanasekaran N, Heasley LE and Johnson GL. (1995). Mahmoud S, Staley J, Taylor J, Bogden A, Moreau J-PL, Endocrine Rev., 16, 259 ± 270. Coy D, Avis I, Cuttitta F, Mulshine JL and Moody TW. Dockray GJ. (1999). J. Physiol., 518, 315 ± 324. (1991). Cancer Res., 51, 1798 ± 1802. Everard MJ, Macaulay VM, Millar JL and Smith IE. (1993). Mao J, Xie W, Yuan H, Simon MI, Mano H and Wu D. Eur. J. Cancer, 29A, 1450 ± 1453. (1998). EMBO J., 17, 5638 ± 5646. Fathi Z, Corjay MH, Shapira H, Wada E, Benya R, Jensen Markowitz S, Krystal G, Lebacq-Perheyden AM, Way J, R, Viallet J, Sausville EA and Battey JF. (1993). J. Biol. Sausville EA and Battey J. (1988). J. Clin. Invest., 82, Chem., 268, 5979 ± 5984. 808 ± 815. Fruhawald MC, O'Dorisio MS, Cottingham SL, Qualman Markwalder R and Reubi JC. (1999). Cancer Res., 59, 1152 ± SJ and O'Dorisio TM. (1998). Ann. New York Acad. Sci., 1159. 865, 420 ± 426.

Oncogene Neuropeptide signaling in cancer LE Heasley 1569 Miller YE. (1989). Am.Rev.Respira.Dis.,140, 283 ± 284. Sethi T, Langdon S, Smyth J and Rozengurt E. (1992). MitchellFM,HeasleyLE,QianN-X,ZamarripaJand Cancer Res., 52, 2737s ± 2742s. Johnson GL. (1995). J. Biol. Chem., 270, 8623 ± 8628. Sethi T, Herget T, Wu SV, Walsh JH and Rozengurt E. Moody TW, Pert CB, Gazdar AF, Carney DN and Minna (1993). Cancer Res., 53, 5208 ± 5213. JD. (1981). Science, 214, 1246 ± 1248. Seu€erlein T and Rozengurt E. (1996). Cancer Res., 56, Moody TW, Carney DN, Korman LY, Gazdar AF and 5758 ± 5764. Minna JD. (1985). Life Sci., 36, 1727 ± 1732. Smith JP, Liu G, Soundararajan V, McLaughlin PJ and Moody TW, Staley J, Zia F, Coy DH and Jensen RT. (1992). Zagon IS. (1994). Am. J. Physiol., 266, R277 ± R283. J. Pharmacol. Exp. Ther., 263, 311 ± 317. Smith JP, Shih A, Wu Y, McLaughlin PJ and Zagon IS. Nagalla SR and Spindel ER. (1994). Cancer Res., 54, 4461 ± (1996). Am. J. Physiol., 270, R1078 ± R1084. 4467. Staley J, Fiskum G and Moody TW. (1989). Biochem. Nagao M, Kaziro Y and Itoh H. (1999). Oncogene, 18, Biophys. Res. Comm., 163, 605 ± 610. 4425 ± 4434. Staley J, Jensen RT and Moody TW. (1990). Peptides, 11, Nelson JB and Carducci MA. (2000). Cancer Invest., 18, 87 ± 1033 ± 1036. 96. Strassheim D, Shafer SH, Phelps SH and Williams CL. Nemeno€ RA. (1998). Front. Biosci., 3, D194 ± D207. (2000). Cancer Res., 60, 2730 ± 2736. North WG. (1991). J. Clin. Endocrinol. Metab., 73, 1316 ± Sun B, Halmos G, Schally AV, Wang X and Martinez M. 1320. (2000a). Prostate, 42, 295 ± 303. North WG, Fay MJ, Longo KA and Du J. (1998). Cancer Sun B, Schally AV and Halmos G. (2000b). Regul. Pept., 90, Res., 58, 1866 ± 1871. 77 ± 84. Nyeki O, Rill A, Schon I, Orosz A, Schrett J, Bartha L and Sundler F, Ekblad E and Hakanson R. (1991). Acta Oncol., Nagy J. (1998). J. Pept. Sci., 4, 486 ± 495. 30, 419 ± 427. Ohki-Hamazaki H. (2000). Prog. Neurobiol., 62, 297 ± 312. Thibonnier M, Berti-Mattera LN, Dulin N, Conarty DM Ohki-Hamazaki H, Watase K, Yamamoto K, Ogura H, and Mattera R. (1998). Prog. Brain Res., 119, 147 ± 161. Yamano M, Yamada K, Maeno H, Imaki J, Kikuyama S, Toi-Scott M, Jones CLA and Kane MA. (1996). Lung Wada E and Wada K. (1997). Nature, 390, 165 ± 169. Cancer, 15, 341 ± 354. Paolucci L and Rozengurt E. (1999). Cancer Res., 59, 572 ± Vara Prasad MVVS, Dermott JM, Heasley LE, Johnson GL 577. and Dhanasekaran N. (1995). J. Biol. Chem., 270, 18655 ± Picon A, Leblond-Francillard M, Ran-Sanson ML, Lenne 18659. F, Bertagna X and de Keyzer Y. (1995). J. Mol. Verbeeck MAE, Elands JPM, de Leij LFMH, Buys CHCM, Endocrinol., 15, 187 ± 194. Carney DN, Bepler G, Roebroeck AJM, Van De Ven Quick MW, Lester HA, Davidson N, Simon MI and Aragay WJM and Burbach JPH. (1992). Pathobiology, 60, 136 ± AM. (1996). J. Biol. Chem., 271, 32021 ± 32027. 142. Reeve JG and Bleehen NM. (1994). Bioc. Biop. Res. Comm., Vincent J-P, Mazella J and Kitabgi P. (1999). Trends 199, 1313 ± 1319. Pharmacol. Sci., 20, 302 ± 309. Reubi JC, Schaer JC and Waser B. (1997). Cancer Res., 57, Wadsworth SJ, Gebauer G, van Rossum GD and Dhanase- 1377 ± 1386. karan N. (1997). J. Biol. Chem., 272, 28829 ± 28832. Rosati R, Adil MR, Ali MA, Eliason J, Orosz A, Sebestyen F Wank SA. (1995). Am. J. Physiol., 269, G628 ± G646. and Kalemkerian GP. (1998). Peptides, 19, 1519 ± 1523. Wank SA. (1998). Am. J. Physiol., 274, G607 ± G613. Sausville E, Carney D and Battey J. (1985). J. Biol. Chem., Watanapa P and Williamson RC. (1993). Br. J. Cancer, 67, 260, 10236 ± 10241. 877 ± 884. Schonwasser DC, Marais RM, Marshall CJ and Parker PJ. Weinberg DS, Ruggeri B, Barber MT, Biswas S, Miknyocki (1998). Mol. Cell. Biol., 18, 790 ± 798. S and Waldman SA. (1997). J. Clin. Invest., 100, 597 ± 603. Seckl MJ, Higgins T, Widmer F and Rozengurt E. (1997). Wittau N, Grosse R, Kalkbrenner F, Gohla A, Schultz G Cancer Res., 57, 51 ± 54. and Gudermann T. (2000). Oncogene, 19, 4199 ± 4209. Seethalakshmi L, Mitra SP, Dobner PR, Menon M and Woll PJ and Rozengurt E. (1988). Proc. Natl. Acad. Sci. Carraway RE. (1997). Prostate, 31, 183 ± 192. USA, 85, 1859 ± 1863. Sehgal I, Powers S, Huntley B, Powis G, Pittelkow M and Woll PJ and Rozengurt E. (1989). Bioc. Biop. Res. Comm., Maihle NJ. (1994). Proc. Natl. Acad. Sci. USA, 91, 4673 ± 164, 66 ± 73. 4677. Woll PJ and Rozengurt E. (1990). Cancer Res., 50, 3968 ± Sethi T and Rozengurt E. (1991). Cancer Res., 51, 3621 ± 3973. 3623. Zugaza JL, Waldron RT, Sinnett-Smith J and Rozengurt E. Sethi T and Rozengurt E. (1992). Cancer Res., 52, 6031 ± (1997). J. Biol. Chem., 272, 23952 ± 23960. 6035.

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