Bombesin-like peptides and cancer

Critical

Reviews

ONCOLOGY/ HEMATOLOGY Critical

ELSEVIER

Reviews

in 0ncology:Hematology

Bombesin-like

23 (1996)

2255238

peptides and cancer

Shaun R. Prestona,*, Glenn V. Miller”,

John N. Primroseb

“Academic Unit of’ Surgery, St. Jumesk lJni~er.~ity Hospital, bAcademic Unit of’ Surgery, Southampton General Hospital, Accepted

27 March

Leeds LS9 7TF, l/K Southampton. tiK

1996

Contents 1.

Bombesin-like peptides 1.1. Discovery and characterisation 1.2. ‘Mammalian bombesins 1.3. Patho-phystological acttons

2.

Bombesin

4.

Hormones and cancer. 4.1. Historical overview 4.2. Current therapeutic regimes 4.2.1. Breast cancer 4.2.2. Prostate cancer 4.2.3. Thyroid cancer 4.2.4. Pituitary tumours 4.2.5. Gastro-enteropancreatic

(BRS-3)

antagonists

229

using

hormone

tumours

analogues

230 230 230 230 230 230 230 230

Bombesin-like peptides and carcinogenesis 5.1. Small cell lung cancer 5.2. Medullary thyroid carcinoma 5.3. Pancreatic carcinoma 5.4. Gastric carcinoma 5.5. Colorectal carcinoma, 5.6. Breast carcinoma 5.7. Prostatic carcinoma. 5.8. Gastrinoma 5.9. Duodenal carcinoma

230 230 231 231 23.3 232 233 231 233 23.1

6.

Discussion

233

7.

Conclusions

234

Reviewers.

235

References

235

author.

lO40-8428/96;S32.00 PII

receptor

(GRP-R)

227 227 228 228 228 228

5.

Biographies.

* Correspo*lding

...

Bomb&n receptors 2.1. Discovery and characterisation 2.2. Cloning the receptor genes 2.2.1. Gastrin Releasing Peptide Receptor 2.2.2. Neuromedin B Receptor (NMB-R) 2.2.3. Bombesin Receptor Subtype Three

3.

225 225 226 321

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K 1996 Elsevier

238

113 2433144; Science

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fax: Ltd.

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1. Bombesin-like 1.1. Discowry

S. R. Prcstm

et rd. II Critical

Reviews

peptides rrnd churucterisution

Amphibian skin is a well recognised source of novel peptides. many of which, upon further investigation, have been found to possess ubiquitous mammalian counterparts: physalaemin, substance P; caerulein, gastrin/cholecystokinin; bradykinin and phyllokinin, bradykinins. Systematic screening of amphibian skin for bioactive peptides led to the discovery of the bombesin-like peptides. Methanol extracts from the skin of three European discoglossid frogs were found to contain the bioactive tetradecapeptides: bombesin from Bombinu bombinu and Bombinu cariegutrr wriegutu and alytesin from Alytes obstetricwzs [l]. Nakajima et al., working simultaneously, identified and characterised a very similar peptide from the skin of RLUIL/ pipicws, the undecapeptide known as ranatensin [2]. The primary sequence of bombesin, alytesin and ranatensin showed considerable homology and the peptides produced almost identical effects in pharmacological bioassays [2,3]. Closer comparison of the primary structures of these and subsequently discovered, related amphibian peptides revealed greatest sequence homology in the C-terminal region, enabling their sub-division into: the bombesins, the ranatensins and the phyllolitorins. The bombesins were characterised by a leucine residue second from the C-terminus: the ranatensins by a phenylalanine residue in the siime position; and the phyllolitorins by a serine residue third from the C-terminus. In pharmacological assays all peptides possessed a hypertensive effect on systemic blood pressure when infused intravenously into dogs, reflected in the naming of the peptide isolated from Ranu pipiens, rana-‘tensin’ [2.3]. As the screening procedure continued it became apparent that the pharmacological effects of the bombesin-like peptides extended beyond the ‘tensin’ effects initially determined. They caused: a. Stimulation ofgastric sccwtion [3]; b. I;‘Nnc.rcutico-hiliar), stimulution [4], pancreatic enzyme secretion, gall bladder contraction and relaxation of the sphincter of Oddi; c. Disruption oj perisftrltic ucfiri/>’ [5] resulting in intestinal stasis; d. Renal efftic,t,s, afferent glomerular artery constriction, activation of the renin-an<;iotensin system [6] and erythropoeitin release [7]; e. l~~~~pothermiu [8]; f: Hyperglycuemiu [9]; g. Sutiety [lo]; and h. Refeuse qf enteric peptides [l l] increasing plaslr a concentrations of glucagon, insulin and pancreatic polypeptide.

The potent biological effects of these amphibian peptides in mammals prompted the search for a ‘mammalianbombesin’. Screening mammalian tissue with antisera to the carboxyl-te!*minal of bombesin revealed the gas-

in Oncologl~Hemutolo~~

23 (1996)

225.-238

trointestinal tract, brain and lung to be sites rich in bombesin-like immunoreactivity [ 12,131. The first ‘mammalian bombesin’ was isolated from porcine non-antral stomach [14]. The carboxyl terminal of this heptacosapeptide possessed striking sequence homology to bombesin and stimulated the release of gastrin in vivo with similar activity to bombesin. The peptide was thus named ‘gastrin releasing peptide’ (GRP). This name perpetuates despite the fact that the peptide possesses numerous other physiological actions in mammals [15]. An additional bombesin-like peptide, with the amino acid sequence of the carboxyl terminal decapeptide of GRP, originally named neuromedin C, subsequently GRP-10, and ultimately GRP,8.27, was found in canine small intestine [ 161, porcine spinal cord [ 171 and a human pulmonary carcinoid tumour [I 81. Little further work has been published on GRP,,.2,. In 1984 the mRNA transcript encoding human GRP was cloned and characterised [19] and the gene later localised to chromosome 18 [20]. Further cellular analysis revealed the presence of a single GRP gene, two differing mRNA molecules and three different GRP pro-hormones, differing in their prevalence within different cellular compartments [2 1,221. The peptide GRP possesses two tryptic cleavage sites that would generate GRP,,.,, (GRP-10) and GRP,,.,, (GRP-14) [13]. The existence of GRP,,-,, in vivo has not been proven to date. Fractionation of acid extracts of porcine spinal cord revealed one fraction which possessed potent uterine stimulation but weak ileal stimulation. This decapeptide possessed sequence homology with the bombesin-like peptides, especially with those of the ranatensin sub-family. Each novel peptide isolated by this screening process was termed neuromedin; because of its homology with the bombesin-like peptides it was named neuromedin B. Ranatensin-like immunoreactivity was found to possess a different distribution to that of GRP within the rat brain [23]. Like GRP additional related neuromedin B peptides have been isolated, neuromedin-30 and neuromedin-32 in extracts from porcine brain and spinal cord [24]. These peptides both contain the neuromedin B sequence. All three peptides possessed potent activity when assayed on rat uterus. but whether these peptides are neuromedin B precursors or distinct bioactive peptides has not been determined. Cloning and characterisation of neuromedin B mRNA [25] and blot hybridisation analysis of human DNA revealed a single copy of the NMB gene. Two human NMB mRNAs were discovered with levels highest in the hypothalamus, stomach and colon. The NMB sequence lies within NMB-32 flanked by basiccleavage sites, similar to GRP. The gene has been localised to chromosome 15 qll [25]. To date no mammalian homologue of the phyllolitorin subfamily has been isolated, but preliminary HPLC experiments indicate the presence of a phyllolitorin-like

peptide in extracts of lymphoblasts from human leukaemic bone marrow [26]. Complementary DNAs (cDNAs) encoding Leu” and Phe8 phyllolitorin have recently been synthesised from mRNA extracted from the skin of Ph~~llomedusa sauvagei [27]. These cDNAs may now be used to screen mammalian genomic libraries and tissues to de1ermine whether mammalian homologues of this subclass of peptide exist and if so where, and in what form. GRP is found at highest concentrations in the pulmonary neuroendocrine cells of the foetal lung, the neurones of the central nervous system and in the gut myenteric plexus. It was initially thought that bombesins were gut hormones, a:sin birds and amphibia they occur in gut endocrine cells 1281; however in mammals they have an exclusively neuronal origin [29]. Bombesin-like immunoreactivity/GRP has been demonstrated in cell bodies within the submucosal and myenteric plexuses, in nerve terminals in the smooth muscle of the small intestine and colon [30,3 11.The distribution of GRP immunoreactivity varies within the gut: highest levels found in the human fundus, antrum, pylorus and pancreas; lower levels being found in the duodenum, jejunum, terminal ileum and colon [31]. In the gastric antrum and pylorus, GRP is found in high concentrations associated with the mucosa [31], attributed to a submucosal plexus [30], as well as in nerve terminals w!thin the muscle layers. Price et al. [31] also report immunoreactivity in the colonic mucosa. Northern blotting of human adult and foetal gastrointestinal tissue using cDNA hybridisation probes revealed highest levels of GRP mRNA in the colon, moderate levels in stomach and lower levels in duodenum, jejunum, ileum and pancreas [19]. Levels in adult tissues were lower than in corresponding foetal tissue, but the relative abu.ndance within each tissue is the same for foetus and adult [ 1.51. The lack of correlation between mRNA and peptide concentrations indicates regulation at a post-transcriptional level. In the respiratory tract GRP is alsc found at high levels in the developing lung, and at lower levels in adult tissue [32]. High concentrations of neuromedin B-like immunoreactivity have been demonstrated, confined to the nerve fibres of the circular muscle coat, in the oesophagus, jejunum and rectum [33]. Most work subsequently performed on NMB is restricted to the central nervous system where its distribution differs from that of GRP [341. 1.3. Putho-,oh~siologi~al

The mechanisms by which GRP causes gastrin release have been intensively studied, but the remaining effects are less well characterised. Further studies have demonstrated that the chronic administration of bombesin to rats results in G-cell hyperplasia in vivo [38] and the demonstration that purified canine antral G-cells possessed high affinity binding sites for GRP provided support for a direct mitogenic effect [39]. Intraperitoneal injection of bombesin for 7 days into rats has been shown to stimulate DNA synthesis and increase the DNA content of gastric oxyntic gland and colonic mucosa [40]. Administration of bombesin to neonatal rats by subcutaneous injection for 6 days [41] was shown to increase the weights of stomach, intestine and pancreas; the heights of fundal and antral mucosae; and the density of parietal cells was significantly increased. The number of G-cells also appeared to be increased but this failed to achieve statistical significance. Electron morphometric analysis of these tissues revealed hyperplasia of duodenal and pancreatic acinar glands. ‘Chronic’ administration of bombesin to adult rats for 1 week by subcutaneous injection also induces gastrin cell proliferation [38]. Ranatensin and the mammalian homologue neuromedin B have been studied in far less detail than the bombesin subfamily of peptides. Ranatensin has fewer biological effects than bombesin, its effects are largely limited to the stimulation of the contraction of smooth muscle [13]. In the rat oesophagus neuromedin B is capable of causing smooth muscle contraction at lower doses than bombesin or GRP [42] and specific neuromedin B-induced smooth muscle contraction has been demonstrated in human and rabbit rectosigmoid colon [431. Neuromedin B is also capable of inducing release of the gastroenteropancreatic hormones gastrin, insulin [44] and enteroglucagon [45], but not glucagon [44] or somatostatin [46]. Pancreatic exocrine secretion can also be stimulated by NMB [47], but all secretory effects require a higher peptide dose than members of the bombesin subfamily and-thus NMB is not a proven physiological endo-, exo-crine stimulant and may be restricted to regulation of gut motility.

2. Bombesin receptors 2.1. Discovery

und characterization

uctions

GRP is also capable of stimulating release of other gastrointestinal hormones; insulin, glucagon, pancreatic polypeptide [35], cholecystokinin [36], somatostatin [ll], motilin, glucose-dependent insulinotropic polypeptide (gastric inhibitory peptide), vasoactive intestinal polypeptide and enteroglucagon [37].

In order to further investigate the direct effects of bombesin upon cells, studies commenced on established cell lines. Bombesin, at nanomolar concentrations, was demonstrated to be mitogenic to the murine fibroblast cell line Swiss 3T3, enhancing DNA synthesis and cellular proliferation [48], GRP acting in an identical manner [49]. Radioligand displacement assays using “‘I-GRP re-

vealed that GRP bound to specific high affinity receptors on Swiss 3T3 cells. GRP/bombesin induced mitogenesis and radioligand binding were all shown to be inhibited in a dose dependent manner by the bombesin antagonist [D-Arg’. D-Pro2, D-Trp7.“, Leu”]substance P. Neuromedin B was capable of stimulating both mitogenesis and inhibiting ‘251-GRP binding in Swiss 3T3 cells, but only at significantly higher doses than bombesin or GRP [491. The second messenger systems activated by the receptor-ligand interaction were then determined. These included enhanced phosphoinositide metabolism, mobilisation of intracellular calcium stores [50]; activation of protein kinase C [51]; and induction of cellular oncogenes c-nr~~ and c-j& [52]. Tyrosine kinase activity after bombesin-receptor interaction has also been reported in Swiss 3T3 cells [53] resulting in the tyrosine phosphorylation of a cluster of proteins [54-561, inhibition of which prevents both c-j& mRNA expression and DNA synthesis [57]. Guanine-nucleotide-bindingprotein (G-protein) bombesin receptor coupling has been demonstrated in Swiss 3T3 cells [58], with N-UY p21 or a functionally related peptide proposed as the putative G-protein [59]. It is now apparent that the GTPase activity of the YLISproto-oncogene product is increased by a GTPase activating protein (GAP). One component of the proteins tyrosine phosphorylated as a result of bombesin-receptor interaction has been recovered in anti-GAP immunoprecipitates from Swiss 3T3 cells [60]. Specific receptors for the bombesin-like peptides had been demonstrated in the central nervous system [61], on pancreatic acinar cells [62], pituitary tumour cells [63] and small cell lung cancer cell lines [64]. All of these receptors bound GRP in preference to NMB. The rat oesophagus was known to possess high levels of neuromedin B-like immunoreactivity and radioligand displacement assays revealed a second bombesin-like peptides receptor, the neuromedin B preferring subtype. Autoradiographic analysis of rat brains revealed the existence of two pharmacologically distinct receptor populations, some areas binding GRP with higher affinity than neuromedin B and other areas possessing reversed relative affinities [65,66], consistent with the two receptors demonstrated within the rat gastrointestinal tract. The specific bombesin receptor antagonist [DPhe”]bombesin,.,,ethylester was demonstrated to bind to the GRP-preferring subtype of receptor with high affinity and is capable of blocking the receptor induced cellular responses [67]; w:hercas the antagonist has little effect on the responses elicited by receptor-ligand interaction at the neuromedin B preferring receptor subtype [68]. The second messenger transduction mechanisms activated by receptor-ligand interaction on the neuromedin B receptor are less well characterised but inositol phosphate generation and an increase in cytosolic calcium. but no increase in cyclic AMP have been demonstrated

upon the rat glioblastoma cell line C-6 [69]. NMB being 50-fold more potent than GRP in producing these responses. The only evidence for the presence of a phyllolitorin receptor subtype demonstrated to date was the elicitation of intracellular calcium mobilisation by the addition of phyllolitorin to cultures of the colon adenocarcinoma cell line WIDs, but not following addition of GRP or neuromedin B [70]. 2.2. Cloning the receptor genes 2.2.1. Gastrin Relemsing Peptide Receptor (GRP-R) In 1990, two research groups, working independently, isolated and characterised cDNA clones of the GRP-receptor from Swiss 3T3 cells [71,72]. Sequence and hydrophobicity analysis revealed the GRP-receptor to be 384 amino acids long, and with seven potential membranespanning domains. Cloning the human GRP-R from placental and peripheral blood genomic libraries, and human small cell lung cancer cell lines [73] revealed it to possess 90% homology with the Swiss 3T3 GRP-R [73] and the gene localised to the X chromosome on the human genome between pll and qll [74]. 2.2.2. Neuromedin B Receptor (NMB-R) The GRP receptor cDNA probe was used under low stringency hybridisation conditions upon a rat oesophageal cDNA library to detect clones of the NMB receptor [75]. Further analysis revealed the receptor to be composed of 390 amino acids and a member of the seven transmembrane domain superfamily of receptors. The human NMB-R, cloned from placental and peripheral blood genomic libraries and human small cell lung cancer cell lines possessed 89% homology with the rat NMB-R [73] and the gene localised to chromosome 6, q21-qter on the human genome [74]. GRP-R and NMB-R cDNA probes have been used to demonstrate that, like the peptide ligands. bombesin receptor subtypes have a distinct, but overlapping, distribution within the central nervous system [76]. 2.2.3. Bombesin Receptor Subtype Three (BRS-3) A third bombesin receptor subtype composed of 399 amino acids was discovered by two research groups, the first using high stringency hybridisation techniques to screen a Guinea pig cDNA library for additional members of the seven transmembrane domain superfamily of receptors [77] and the second using low stringency Southern blot analysis of a human cDNA library with radiolabelled GRP-R and NMB-R probes [78]. When expressed in cells, radioligand displacement assays revealed bombesin binding sites with low affinity for GRP and extremely low affinity for neuromedin B, differing from both previously described receptors, and patch clamp analysis revealed bombesin, GRP, neu-

romedin B, ranatensin and Phex-phyllolitorin evoked responses at micromolar concentrations. BRS-3 expression was demonstrated within the pregnant guinea pig uterus. rat testis and in small cell, non-small cell and carcinoid lung tumour cells, and at low levels in the brain and the gene localised to the human X chromosome.

3. Bombesin receptor antagonists Antibombesin antibodies were the first molecules used to inhibit the effects of bombesin in experimental systems. but the use of these large macromolecules does not permit pharmacological analysis of the effects, and the potential therapeutic options are severely limited by the development of antibodies to the anti-bombesin antibodies combined with the high risk of anaphylaxis in vivo. The need for potent, competitive. high affinity bombesin receptor antagonists was essential in the further study of the endogenous bombesin-like peptides to enable quantitative investigation of short- and longterm inhibition of their effects. The first Istep in the search for bombesin receptor antagonists came in 1984 when Jensen and co-workers noted that D-amino acid substituted analogues of substance P, known to act as substance P receptor antagonists. were also capable of binding to the bombesin receptor and function as receptor antagonists [79]. These peptides only possessed low affinity for bombesin receptors and their lack of specificity severely limited their usefulness. The next class of antagonists developed were produced from bombesin by D-amino acid substitution for His” [WI. These peptides were more selective but possessed a very low affinity for the receptor. The extremely poor affinity and poor solubility of some antagonists from this class also rendered them inadequate. A new design strategy was employed in the development of the third class of bombesin receptor antagonists, the peptide bond rather than the amino acid side chain or stereo-isomer was altered. In these peptides the CONH peptide bond was changed to either CH,NH (known as a $-bond replacement or a reduced peptide bond) or CH,O (an ether linkage) producing antagonists containing pseudopeptide bonds. The first antagonist with a potency high enough to be useful was the pseudotetradecapeptide [Leu’j. $13-14]bombesin, a peptide in which the CONH bond between amino acids 13 and 14 was replaced with a CH,NH group [81]. Replacement of the 26-27 CONH bond of GRP,,,.,, with an ether linkage also produced the potent antagonist AC-GRP(20-27) [82]. The effect of removing the N-terminal amino acids was then evaluated and resulted in a marked loss of receptor affinity. Excision of

the N-terminal five amino acids from [Leu’“, $13I4lbombesin resulting in [Leu”, t/13- 14lbombesin (614) produced a marked reduction in receptor affinity when binding was assayed upon Swiss 3T3 cells (K, fell from 73 nM to 291 nM). This effect could however be reversed and the receptor affinity even enhanced by the presence of a D-aromatic amino acid in the 6 position; for example, the receptor affinity of [DPhe”, LeulJ. (//13-14lbombesin (6-14) when assayed in the same binding assay upon Swiss 3T3 cells was markedly increased (K, = 7 nM) [83]. Modification of the C-terminus of peptides to produce antagonists was originally devised by Martinez and Bali to produce gastrin antagonists [84] and was applied to bombesin and GRP. In this process the C-terminal methionine residue was removed from the peptides bombesin (6-14) and GRP (30-27) which initially resulted in good competitive antagonists, their potencies enhanced by adding alkyl substitutes to the C-terminal amide moiety. Other C-terminal groups have also resulted in very high affinity antagonists. hydrazines and alkylesters [83]. Until very recently all potent bombesin receptor antagonists possessed high affinity for GRP receptors, but had very low affinity for the neuromedin B receptor. The best neuromedin B antagonist previously had been [DPhe”]bombesin [83]. The first selective neuromedin B receptor antagonist has recently been synthesised [85]. Neuromedin B pseudopeptides, des-Met”‘-NMB, or des-Met”‘-NMB esters, have been synthesised but none of these synthetic pseudopeptide procedures has yielded NMB-R antagonists [85]. Studies have demonstrated that somatostatin and its analogues are capable of inhibiting “‘I-GRP binding to a 120-kDa protein in extracts of Swiss 3T3 cells, and could inhibit binding of opiates to appropriate opiate receptors [86,87]. The ability of somatostatin-14 and -28, and various cyclic somatostatin octapeptide analogues to function as NMB-R and GRP-R antagonists was investigated [85]. They discovered that somatostatin analogues but not native somatostatin-14 or -28 inhibited binding to NMB receptors. The most potent analogue D-Nal-CysTyr-D-Trp-Lys-Val-Cys-Nat-NH, inhibited binding ol ‘251-[DTyr0]NMB to C6 rat glioblastoma cells and NMB receptor-transfected Balb 3T3 cells. K, = 59 nM and 885 nM, respectively. The antagonist had a IOOfold lower affinity for the GRP receptor The octapeptides did not possess agonist activity as assessed by inositol mobilisation, but inhibited NMB stimulated mobilisation. Comparative structural studies of the ability of these octapeptide analogues to antagonise somatostatin. I[-opioid and NMB receptor binding was performed and the exact structural requirements were specific for each receptor suggesting that further modification of these somatostatin analogues may yield more potent selective NMB-R antagonists.

230

4. Hormones

and cancer

4.1. Historicd

orervie\t~

The concept of hormonal manipulation of cancer growth was first suggested in 1896 by Beatson who noted that the ovary ‘held control’ over the breast in the ‘absence of distinct nervous control’, now known to be endocrine in nature. In support of this he demonstrated a dramatic reduction of breast tumour and axillary node volume in a female patient with advanced breast carcinoma following bilateral salpingo-oophorectomy [88]. The importance of sex hormones in relation to prostatic carcinoma was realised earlier this century. In 1941, Huggins and Hodges reported a decrease in serum levels Iof acid phosphatase in patients with metastatic prostate carcinoma when treated with the oestrogen stilbestrol, or following bilateral orchidectomy [89]. 4.2. C’wrent u11ul0gue.s

tkzpeutic

regimes using hormone

4.24. Pituitary turnours Somatostatin analogues have been reported to improve symptoms of patients with functional growth hormone secreting pituitary tumours but to also reduce tumour volume. These analogues may be of benefit in acromegalic patients, especially those who do not benefit from surgery or radiotherapy [96]. 4.:?.5. Gustro-enteropancreatic tumours Somatostatin analogues have been used in functional gastro-enteropancreatic tumours producing symptomatic improvement and tumour regression with gastrinoma [97], VIPoma [98,99] and metastatic carcinoid tumours [loo].

5. Bombesin-like

peptides and carcinogenesis

In addition to their mitogenic effects on Swiss 3T3 fibroblasts the bombesin-like peptides have been implicaied in the pathogenesis of a number of mammalian carcinomas. 5. I. Small cell lung cuncer

The concept of hormone dependent tumour growth is now widely accepted and is used routinely in the management of breast. prostate and thyroid carcinoma. 4.2.1. Breast c‘rmt’er Oestrogen receptor antagonists provided oestrogen antagonism without oophorectomy or adrenalectomy. LJsed as an adjunct to surgery tamoxifen has resulted in prolongation of disease-free survival in both pre- and post-menopausal women with breast carcinoma [90]. The progestins megestrol and medroxyprogesterone acetate have also been shown to induce tumour regression in advanced breast carcinoma [91]. GnRH agonist trials have reported 45% objective response rates in unselected patients and 49% in women with oestrogen reccptor positive tumours [92]. 4.22. Prostutr cancer As an alternative to bilateral orchidectomy LHRH analogues are also used to block pituitary LHRH receptors and decrease testicular androgen production to castration levels in metastatic prostate carcinoma [93]. 4.2.3. Thyroid cunccr The treatment of metastatic deposits from a well differentiated thyroid carcinoma with L-thyroxine were first described in 1954. It is now thought that most differentiated thyroid carcinomas, papillary and follicular, are TSH-dependent [94] and thyroxine is recommended even if total thyroidectomy has not been performed [95].

Bombesin-like immunoreactivity (BLI) is known to be expressed within human lung tissue at high concentrations in the foetal/neonatal lung [32] but at much lower concentrations in adults [lOl]. Bombesin and GRP have also been demonstrated to be growth factors for normal human bronchial epithelial cells when assessed in vitro using a clonagenic assay [102]. Small cell lung cancer (SCLC) cells are known to contain large numbers of neurosecretory granules. For this reason Moody and co-workers screened 17 SCLC, and eight non-SCLC culture lines for the presence of bombesin immunoreactivity. They discovered that protein extracts from all SCLC lines tested contained bombesin-like immunoreactivity at varying levels, but none was detected in the non-SCLC cells [103]. A number of human SCLC tumours were also demonstrated to contain high levels of BLI [104]. The SCLC cells were then demonstrated to secrete bombesin-like peptide into the surrounding media and HPLC of this peptide revealed it to differ from synthetic bombesin [105]. Bombesin was then demonstrated to be a potent mitogen to SCLC [106], and this was subsequently demonstrated with GRP [107]. This effect was demonstrable on SCLC but not lung squamous cell or adenocarcinoma. A single class of high affinity binding sites for bombesin-like peptides was subsequently demonstrated upon these human SCLC cell lines [108]. These findings suggested that bombesin/GRP acted as an autocrine growth factor. Cuttitta and co-workers developed a murine monoclonal antibody. 2A11, to Lys3-bombesin a synthetic

bombesin-like peptide with C-terminal homology with human GRP. This antibody was found to inhibit hormone receptor interaction, and inhibited clonal growth of SCLC in vitro and the growth of SCLC xenografts in vivo using nude mice [64]. These effects have also been demonstrated using high affinity [Psi”.‘4]-bombesin/GRP antagonists [ 1091. The murine monoclonal anti-GRP/bombesin antibody 2A11 has been used in a Phase II trial upon 12 human subjects with small cell lung cancer; patients received a 4-week course of 2All. This treatment resulted in a complete response in one patient, documented on chest X-ray, chest CT scan and by direct bronchoscopic visualisation, stable disease in four patients. six had progressive disease and one could not be assessed. The completely responding patient, who had a SCLC recurrence in her lung, maintained a complete response for 5 months, following which tumour was visualised bronchoscopically at the initial site of recurrence. This lumour recurrence progressed despite further treatment with 2All. Following three 4-week courses of 2.411 the completely responding patient did not develop human anti-mouse antibodies to the 2A11 antibody. The exact mechanism by which this tumour response occurred is unclear as the patients tumour biopsies did not reveal GRP staining [l lo]. Using semi-solid medium clonagenic assay neuromedin B has also been demonstrated to stimulate growth of SCLC lines in vitro over the concentration range 3-30 nM [ill].

Ectopic hormone production is common in medullary carcinoma of the thyroid as well as in small cell lung cancer. Immunoreactivity to GRP has been demonstrated at high concentrations in medullary carcinomas [15,112]. Further gel filtration analysis of the immunoreactivity found it to be composed of two components. corresponding to GRP,.,, and GRP 14.27 [ 1121. GRP immunoreactivity within calcitonin-releasing cell (C.cell) in normal thyroid tissue has been demonstrated to vary with age peaking at 2-2.5 months, the mean GRP-like immunoreactivity in 3- to 20-week-old infants being 20 times greater than the mean level in normal adults [15]. Elevated levels of GRP expression have also been demonstrated in C-cell hyperplasia in a patient with hypercalcaemia [15]. Using in situ hybridisation and immunoperoxidase analysis it has been demonstrated that the majority of neonatal C-cells express GRP, but this decreases to 5 -- 18% in normal adults. The level of GRP expression was examined in C-cells adjacent to follicular adenomas and papillary carcinomas and the proportion of GRPpositive cells was greatly elevated to 45-70’%1 [15]. These data suggest a potential role for GRP in the

normal neonatal thyroid development and also in C-cell hyperplasia, C-cell neoplasia (medullary carcinoma) and as a paracrine growth factor for non-neuroendocrine thyroid carcinomas.

Chronic treatment with bombesin has been demon:strated to increase the DNA content of the pancreas 11131. Lhoste and Longnecker reported that treatment of rats with bombesin stimulates the growth of azaserme induced atypical acinar cell nodules, recognised preneoplastic acinar cell lesions of the pancreas. In (addition they noted that bombesin treatment alone resulted in an increase in pancreatic weight and concluded that bombesin stimulated the growth of normal and induced preneoplastic pancreatic acinar cells and was thus thought to function as a tumour promoter [114]. Bombesin, administered by intraperitoneal injection three times each day for 14 days, resulted in significant pancreatic hyperplasia, with an increase in pancreatic weight, protein, and DNA content. Concomitant treatment with a cholecystokinin (CCK) antagonist only inhibited bombesin-stimulated DNA content. The trophic actions of bombesin on the pancreas are therefore thought to occur both directly and indirectly through CCK. It was also noted that bombesin induced biosynthesis of the polyamine putrescine. but not spermine or spermidine, within the rat pancreas as early as 2 h after administration. This effect was unaffected by CCK antagonist treatment and is proposed as the mechanism by which bombesin exerts its direct trophic effect upon the rat pancreas [115]. Hamsters, pre-treated with the pancreatic carcinogen N-nitrosobis(2-oxopropyl)amine (BOP), followed by 2 months treatment with the high affinity bombesin receptor antagonist RC-3095 demonstrated a significant reduction in the number of hamsters developing pancreatic cancers and the number of tumour nodules per animal. Analysis of the tumours resected from the hamster pancreas revealed them to express high affinity bombesin receptors [116]. In a similar study, RC-3095 was demonstrated to inhibit growth of BOP induced tumours, but tumour growth *as also inhibited by administration of GRP. The concomitant administration of bombesin and GRP(14-27) did not nullify the inhibitory effect demonstrated by RC-3095. These data suggest that the effect of these peptides on pancreatic cancer is more complex than was initially thought [ 1171. The human pancreatic cancer cell line CFPAC-1 has been demonstrated to express high affinity binding sites for bombesin and growth inhibition has been demonstrated in vivo, using a nude mouse xenograft model, and in vitro, monitoring [iH]thymidine incorporation, by treatment with RC-3095. Bombesin stimulated DNA synthesis has been demonstrated in these cells and

concurrent treatment with RC-3095 abolished this effect, RC-3095 appearing to inhibit growth of CFPACI cells by blocking the interaction of bombesin-like peptides with their receptors [118].

Following the discovery that bombesin could stimulate the development of preneoplastic lesions in the pancreas, Tatsua and co-workers investigated the effect of bombesin on gastric carcinogenesis induced by !V-methyl-N’-nitro-N-nitrosoguanidine (MNNG) in rats. They demonstrated that prolonged administration of bombesin at 20 /ig/:kg, administered subcutaneously on alternate days, significantly increased the number of gastric cancers per rat, but at 40 ,ugg/kg the incidence, and number per rat, of gastric cancers were significantly increased. Bombesin administration did not however aiter the histological appearance or their depth of tumour invasion [119]. The bombesrn induced enhancement of the MNNG chemical gastric carcinogenesis model is thought to occur at least in part by an increase in polyamine biosynthesis. As mentioned above bombesin rapidly increases polyamine synthesis in the pancreas, the ratelimiting enzyme for this process being ornithine decarboxylase (ODC). In rats treated with MNNG administration of bombesin was also noted to increase the ODC activity and the bromodeoxyuridine labelling index of the gastric antrum. The administration of the ODC inhibitor 1,3-diaminopropane with bombesin, following MNNG. reduced the bombesin enhancement of gastric cancer back to control levels [120]. The presence of single site high affinity binding sites for GRP on human gastric cancer tissue has previously been described by ourselves. Of 23 gastric cancers examined, 57% expressed GRP binding sites which were also present upon one patient’s gastric mucosa who had the hyperproliferative, premalignant syndrome of Men&trier’s disease. GRP binding sites were not however demonstrated upon any of the uninvolved gastric mucosa examined from the same 23 patients [ 1211. We have also described the presence of functional GRP receptors upon the human gastric cancer cell line St42, which will provide a useful model for future studies [122]. Recently one group have demonstrated growth inhibition of nude mouse xenografts of the human gastric cancer cell line MKN45 following treatment with the high affinity bombesin/GRP receptor antagonist [DTpi”,Leu” tl/(CHzNH)Leu’4]-bombesin(6-14) (RC3095) [123]. Daily treatment with 20 ,ug/day of RC3095 resulted in inhibition of tumour growth which became highly ‘significant at 2 weeks, and tumour doubling time was increased from Il.3 days in the control group to 32.9 days in the group treated with RC-3095.

The mechanism of this inhibition is unknown. The same group have also characterised the presence of GRP-Rs on the human gastric cancer cell line Hs746T and demonstrated increased DNA synthesis in vitro in response to bombesin, an effect which was inhibited by the receptor antagonist RC-3095. Growth of this tumour cell line in vivo, grown as xenografts in athymic nude mice, was also significantly inhibited by treatment with RC-3095 at 20 ,ugg/day for 21 days, tumour doubling time increased from 3.6 to 5.1 days [124]. 5.5. Colorrctal

carcinoma

The stimulation of rat colonic mucosal DNA synthesis by BBS has already been noted above [40]. In 1990, Narayan and colleagues demonstrated the presence of two classes of high affinity binding sites for GRP on the murine colon cancer cell line MC-26. Using [“Hlthymidine and 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide uptake as in vitro assays they also demonstrated that bombesin over the concentration range 0.5550 nM could elicit significant dose-dependent growth effects upon the MC-26 cells [125]. This group also demonstrated the presence of a single class of high affinity GRP binding site upon membrane preparations of murine colonic mucosa [126]. The growth of the human colon cancer cell line HT29. when grown as xenografts in nude mice has been demonstrated to be significantly inhibited by treatment of the mice with subcutaneous RC-3095 at 20 ,ug/day. Inhibition of tumour growth was highly significant from 21 days following commencement of treatment and tumour doubling time was increased from 4.1 days in the control group to 7.2 days with treatment [127]. The GRP-receptor status of this cell line was not however confirmed in this study. High affinity bombesin binding sites were subsequently demonstrated on membrane preparations from resected tissue from six of 15 human colon cancers. but not on uninvolved colonic mucosa [128]. We have also confirmed these findings and demonstrated that the binding sites expressed on colorectal cancer tissue are of the GRP preferring subtype [129]. In a subsequent study we also described the presence of high affinity GRP binding sites on the human colonic cancer cell line Colo320HSR [130], which have subsequently been shown to be functional receptors [131]. Inhibition of the growth of Colo320HSR by the GRP receptor antagonist has also been demonstrated in vivo using a clonagenic assay [130]. Mobilisation of intracellular calcium has also been demonstrated in LoVo and HCTl16 human colon cancer cell lines [131].

As a result of the detection of bombesin-like immunoreactivity in human and animal milk, human breast epithelial and cancer cells were examined for the presence of binding sites for bombesin-like peptides. Eight breast cancer cell lines and two long-term cultures of breast epithelial cells were screened; three cancer cell lines expressed single, high affinity binding sites for CJRP not expressed by the other cancer cell lines or the epithelial cells in culture [132]. The cells did not contain mRNA transcripts for GRP or NMB and bombesin could not elicit demonstrable growth effects. In another study however immunoreactivity to GRP,, 27 was demonstrated in 16 of 41 human breast cancer biopsies, and GRP,,.?, stimulation of [‘Hlthymidine incorporation was demonstrated on T47D, a GRP receptor expressing cell line [133]. Bombesin has been demonstrated to stimulate growth of the human breast cancer cell line MCF-7 MIII, when assayed in vitro using [‘Hlthymidine incorporation, an effect which could be suppressed by RC3095 [134]. In vivo growth inhibition of this cell line by RC-3095 was also demonstrated when grown as xenografts in nude mice [135].

Bombesin, over the concentration range O.l- 10 nM, has been demonstrated in vitro to stimulate growth of PC-3, a human prostatic cancer cell line demonstrated to possess high affinity binding sites for GRP. Antibodies to GRP were demonstrated to inhibit this growth response [136]. In vivo inhibition of the growth of the human prostatic cancer cell line PC-82, grown as xenografts in nude mice, has been demonstrated using the bombesin receptor antagonist RC-3095 at a dose of 20 ,/ig;day administered by subcutaneous injection. Significant inhibition of tumour growth was observed 3 weeks after commencing treatment and tumour doubling time increased from 11 .O days to 35.4 days in animals treated with RC-3035 [137]. The bombesin-like peptide receptor status of the PC-82 cells was unfortunately not reported and thus the mechanism by which tumour inhibition is likely to have occurred is uncertain. In an almost identical experiment performed upon the PC-3 human prostatic carcinoma cell line, RC-3095 significantly inhibited tumour growth and increased tumour doubling time from 9.2 days to 14.1 days [138].

When administered to rats, plasia of the gastrin releasing gastric antral mucosa [38]. demonstrated the presence of

bombesin caused hypercells (G-cells) within the One subsequent study high affinity GRP bind-

ing sites on canine antral gastrin cells, suggesting the possibility of a direct stimulatory effect [39]. Bombesin has also been demonstrated to stimulate growth of a cell line derived from a functioning human gastrinoma nn vivo grown as xenografts in nude mice. Bombesin treatment resulted in a decrease in tumour doubling time from 20 days to 9 days [139]. GRP is known to be Isecreted from nerve terminals in the gastric wall and pancreas and thus may act as a growth promoter to the neoplastic cells, directly or indirectly. but not by an autocrine loop as these cells do not secrete bombesinlike peptides.

The epithelial cell line HuTu-80. derived from a human duodenal adenocarcinoma, has recently been shown to possess single site, high-affinity, functional GRP-receptors. The cell line does not secrete bombesinlike peptides and despite receptor-ligand interaction initiating inositol phosphate formation no growth effects were demonstrated [140].

6. Discussion The bombesin-like peptides occur widely throughout the body predominantly within the brain, lung and gastrointestinal tract. The peripheral role of GRP is well documented and appears to be involved in foetal/ neonatal lung and neonatal thyroid C-cell development, to regulate the release of a wide number of entero-pancreatic hormones within the gastrointestinal tract and to regulate gut motility. The Feyrter cells within the lung and the C-cells within the thyroid gland both belong to the amine precursor uptake decarboxylation (APUD) system of cells, and may upon malignant transformation progress to small cell lung cancer and medullary thyroid carcinoma, respectively. These tumours are known to secrete GRP which may function as an autocrineiparacrine growth factor. NMB has not been studied as thoroughly and consequently can only confidently be stated to regulate gut motility by NMBRs distinct from those to GRP, but may also be a mitogenic peptide in a number of SCLCs, being mitogenie in vitro and some SCLC cell lines are known to express functional NMB-Rs. With the recent development of NMB antagonists the in vivo effects resulting from NMB antagonism should soon be determined. The existence of mammalian phyllolitorins will hopefully be confirmed or refuted following the development of cDNA probes and the function of BRS-3 may be determined. The following discussion will however concentrate upon GRP. GRP is a well established mitogen to the murine fibroblast cell line Swiss 3T3. Its role as an autocrine

growth factor for SCLC has been recognised in vitro and the recent clinical trial report that the monoclonal anti-bombesin antibody 2A11 induced a clinical response appears to offer a new therapeutic option in the treatment of 1his disease which carries an extremely poor prognosis [I IO]. Potent GRP-R antagonists are already available and may offer an alternative therapeutic tool to determine the impact of GRP antagonism ill SCLC. GRP has been implicated in the neoplastic process of a wide variety of cancers. The effect upon the tumour appears to be :umour promotion. This effect may be a direct result of’ GRP-receptor interaction upon the cell surface or indirect, due to release of other trophic peptides from nearby paracrine cells at which GRP-receptor interaction occurs. Even when the trophic effect is direct the mechanism by which tumour promotion occurs does not appear to be constant. In some tumours. SCLC and medullary thyroid carcinoma, there is ectopic GRP production which may act in an autocrine manner. In the thyroid however the increase in GRP expression occurs in C-cells adjacent to follicular adenomas and papillary carcinomas, with the potential to act as ectopic paracrine trophic factors to the tumour cells. In ather tissues there appears to be overexpression of tke GRP-R above that in surrounding non-cancerous tissue (gastric, colon, breast). Expression or overexpression of GRP-Rs in cancer cells may result in an increaseti. sensitivity of these cells to the existing levels of GRP. a potential mitogenic stimulus, within the local environment. GRP may exert its effects over much greater distances than classical neurotransmitters as they diffuse several micrometers through the mucosa to exert their effects on the G-cells. This fact has been demonstrated as the proportion of G-cells found within a distance of 2 /urn from bombesin-like immunoreactive nerve tibres uas found to be low in all mammals (guinea-pig. 6%: rat. 72%; dog, 14% and human, 9%) [ 1411. Thus cells need not be directly innervated by peptidergic neurones to be influenced by their transmitters. Comparison of the nucleotide sequences of human GRP-R and NMB-R from several human small cell lung cancer cell lines with those from the normal gcnome revealed no difference for each subtype [73] suggesting that the bombesin-like peptide dependent growth observed in cancer cells does not require a structural change within the receptor. The ability c,f GRP to cause release of a number of other enteropancreatic hormones is well established. Many of these peptides have potential mitogenic effects themselves an<: thus GRP may provide a mitogenic stimulus indirectly through other peptide hormones. The interaction between GRP and other peptides with mitogenic potential may also occur at the level of the cell membrane by receptor modulation. GRP has been

demonstrated to upregulate EGF receptors in human pancreatic cancer cells [142] and to downregulate EGF receptors in Swiss 3T3 cells [51]. Ligand-receptor interaction may thus result in activation of a Ser/Thr kinase from the GRP-receptor and a potent Tyr kinase from the upregulated EGF receptor, a combination which is essential for activating the cascade of ‘switch kinases’ some of which require dual phosphorylation for full activation e.g. MAP kinases which may be the ‘site of convergence’ regulating cell division [143]. The benefits of inhibiting the effects of bombesin-like peptides in the treatment of cancer are yet to be determined. The methods which may potentially be used to determine these effects are: (i) Receptor antagonism. The availability of high aflinity GRP-R antagonists, and more recently a NMBR antagonist, provides a simple means of inhibiting the potential mitogenic stimulus. This may be used alone or in combination with other peptide analogues, receptor agonists or antagonists, to inhibit the effects of other potential mitogens released in response to GRP. (ii) Antisense oligonucleotides. As recombinant DNA technology increases, gene therapy may become a more common therapeutic option, the direct inhibition of GRPIGRP-R. NMBINMB-R, mRNA translation may be achieved at a cellular level. This technique has already been demonstrated in vitro in an extremely elegant series of experiments performed on human and rabbit colonic smooth muscle to demonstrate the presence of both GRP-R and NMB-R receptor subtypes. This was achieved by the selective inhibition of smooth muscle contraction in response to each peptide using GRP-R and NMB-R antisense oligonucleotides [43]. This may be expanded to become a therapeutic tool in the treatment of cancer.

7. Conclusions The bombesin-like peptides and their receptor expression appear to be implicated in the pathogenesis of cancer growth in a wide variety of mammalian tissues. The peptide GRP is established as an autocrine growth factor in small cell lung cancer and appears to act as a paracrine growth factor in thyroid tumours, but the exact role of this family of peptides in the growth regulation of most cancers is still to be determined. In small cell lung cancer, the first cancer in which the mitogenic potential of GRP was realised, and one which currently carries a very poor prognosis, the use of GRP antagonism as a true therapeutic option is being established with the commencement of clinical trials, the first report of which has been discussed above. The role of the bombesin-like peptides in tumour growth regulation warrants further investigation and as more detailed knowledge of its effects is pub-

S.R.

Preston

et al. : Critical

Reoielcx

lished, further therapeutic options may evolve and become established in cancer therapy.

in Oncr)lofi?,!Henlutoloec’

[IS]

[19]

Reviewers [20]

This paper was reviewed by G.R. Poston, Clinical Director, Royal Liverpool and Broadgreen University Hospitals NHS Trust, Liverpool, UK, and R. Daniel Beauchamp, MD, Department of Surgery, Vanderbilt University. Nashville, Tennessee, USA.

[21]

[22]

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Biographies Shaun R. Preston BSc, MB, FRCS is Research Fellow in Surgery, St. James’s University Hospital, Leeds, UK. Glenn I/. Miller MB, FRCS is Research Fellow in Surgery, St. James’s University Hospital, Leeds, UK. John N. Primrose MD, FRCS is Professor of Surgery, Southampton General Hospital, Southampton, UK.