(CANCER RESEARCH 53. 5(190-5092, November 1. 1993]

Advances in Brief

High Affinity Binding Sites for Releasing on Human Gastric Cancer and Ménétrier'sMucosa1

Shaun R. Preston,2-3 Linda F. Woodhouse, Steven Jones-Blackett,4 Judy I. Wyatt, and John N. Primrose

Academic Unit of Surgery ¡S.R. P., L. F. W., S. J-B., J. N. P.] and Department of Pathology ¡J.I. Vf.], St. James's university Hospital, Leeds, LS9 7TF, United Kingdom

Abstract creas (reviewed in Ref. 7), and have been shown to promote chemi cally induced gastric cancer in rats (10). In addition, these are The -like peptides gastrin releasing peptide (GRP) and neu- known to be important in the autocrine growth of a number of human romedin B are found in the submucosal and myenteric plexuses of the small cell lung cancer cell lines (11) and have been implicated in the human . These peptides are potent mitogens to Swiss 3T3 fibroblasts and are important autocrine growth factors in human pathogenesis of other solid malignancies, for example colon cancer small cell lung cancer cells. We have recently described the presence of (12). Previous work in our laboratory demonstrated bombesin recep receptors for the bombesin-like peptide, GRP, on the human gastric can tors of the GRP-preferring subtype on a human gastric cancer cell line, cer cell line St42. In this study, we examined fresh resected gastric cancer St42 (13). In this study we have investigated human gastric cancer and uninvolved mucosa from 23 patients for the presence of binding sites tissue and gastric mucosa for the presence of high affinity binding to the bombesin-like peptides. Thirteen of 23 gastric cancers expressed sites for bombesin-like peptides. high affinity binding sites for bombesin (mean K(l = 3.42 IIM:mean H,,,.,x = 40.5 pmol/mg protein), of which 12 were subsequently characterized and Materials and Methods found to be of the GRP-preferring subtype. One of 23 mucosa! samples specifically bound bombesin and was the only sample from a patient with Tissue Collection Ménétrier'sdisease,a disorder of mucosal growth known to be premalig- Immediately upon excision of the stomach for histologically proven gastric nant. The early gastric cancer from this patient also possessed high affinity adenocarcinoma approximately 50% of the tumor was removed, ensuring that binding sites for GRP. This is the first description of binding sites to bombesin-like peptides on human gastric cancer and Ménétrier'smucosa. the edge of the tumor was left intact and that the full thickness of the tumor was not removed. A strip of macroscopically normal gastric mucosa, from the The role of bombesin/GRP antagonists in the treatment of gastric cancer corpus or antrum, distant from the tumor was dissected free from the under warrants investigation. lying submucosa. Small samples of both cancer and mucosa were sent for Introduction routine histology. The remainder of the excised tumor and mucosa were then cut into small pieces and snap-frozen in liquid nitrogen. The specimens were Gastric cancer is estimated to be the second most common cancer then stored at -70°C until required. in the world (1). In the United Kingdom it is the fourth most common cause of cancer death in both men and women, with an incidence of Tissue Processing 270 cases per million population in men and 167 cases per million population in women, diagnosed each year (2). The pathogenesis of Tissue samples, all less than 1 year old, stored as above, were powdered in liquid nitrogen using a department-made piston gun. Powdered tissue not used gastric cancer remains elusive and therapeutic options limited. Bom immediately was restored in sealed containers at -70°C for subsequent use. besin is a peptide originally extracted from the skin of the European discoglossid frog Bombina bombino (3) and possessed profound bio logical potency in mammals. This led to the discovery of the "mam Membrane Preparation malian bombesins" GRP5 (4) and NMB (5). In the human gastroin To assay for the presence of bombesin receptors 0.2 g of powdered tissue testinal tract GRP is found at highest concentrations in the fundus, was taken and placed in 12 ml of ice cold homogenization buffer (300 HIM antrum, and pylorus of the stomach and in the pancreas. Three recep sucrose-1 HIM ethylene glycol-bis(ß-aminoethyl ether)N,N,N',N'-tetraacetic tors for the bombesin-like peptides have been described and cloned: acid- 100 Kallikrein inhibiting units/ml aprotinin-25 mMTris, pH 7.5) and then the GRP-preferring subtype (GRP receptor) (6), found in the gut from homogenized, on ice, for 5 x 5 s at maximum speed using an Ultraturax T25 the esophagus to the rectum (reviewed in Ref. 7); the NMB-preferring homogenizer (Janke and Kunkel, The Scientific Instrument Centre Limited, subtype (NMB receptor) (8), present in the esophageal muscularis Liverpool, United Kingdom). The homogenate was then centrifuged at 500 g for 10 min at 4°Cin a Mistral 3000E MSE centrifuge to precipitate any tissue mucosa (5); and bombesin receptor subtype 3 present in the testis and lung cancer (small cell lung cancer and non-small cell lung cancer) debris, undisrupted cells, and cell nuclei. The supernatant was removed and centrifuged at 70,000 g for 45 min at 4°Cin a Beckman L5-65B ultracentri- cell lines, but not in the rat gastrointestinal tract (9). In the gut GRP fuge to pellet the membrane fraction. The resulting pellet was resuspended in and NMB regulate gut motility, cause secretion of large numbers of 150-300 (¿Imembrane buffer (50 mM 4-(2-hydroxyethyl)-l-piperazineethane- enteric peptide hormones, stimulate exocrine secretion from the pan- sulfonic acid-KOH buffer, pH 7.4, plus 10 mM MgCl2, 0.01% bacitracin, 200 Kallikrein inhibiting units/ml aprotinin) and the protein concentration deter mined by the method of Bradford (14). The protein concentration of the Received 8/9/93; accepted 9/17/93. The cosls of publication of this article were defrayed in part by the payment of page resulting membrane preparation was then adjusted using membrane buffer to a charges. This article must therefore be hereby marked advertisement in accordance with standard concentration of 5 mg/ml. All membranes were prepared fresh for use 18 U.S.C. Section 1734 solely to indicate this fact. the same day. 1Supported by Grant L232 from the Yorkshire Cancer Research Campaign, Harrogate, United Kingdom. 2 Funded by Glaxo Group Research, Ltd.. Greenford, United Kingdom. ' To whom requests for reprints should be addressed. Binding Assays 4 Funded by the Yorkshire Cancer Research Campaign. s The abbreviations used are: GRP. gastrin releasing peptide; BBS. bombesin; EGF. Screening Tissue for Bombesin Binding Sites. One hundred /1u of tumor/ mucosal membrane protein, prepared as above, were incubated with 125I-Tyr4- epidermal growth factor; ICso, mean inhibitory concentration; NMB neuromedin B; TGFa, transforming growth factor a. bombesin (approximately 200 pM; Du Pont Ltd., Stevenage, United Kingdom) 5090

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1993 American Association for Cancer Research. OASTRIN RELEASING PEPTIDE BINDING SITES ON HUMAN GASTRIC CANCER alone (total bound) or with 1 X 10 6 Mbombesin (Bachern United Kingdom, Table 2 Histological data on uninvolved gastric mucosa Ltd., Saffron Waiden, United Kingdom) (nonspecific bound) in incubation buffer (membrane buffer plus 40 fig/ml bestatin and 0.02% bovine serum albumin) in a reaction volume of 100 /xl, with each point performed in sex- tuplets. The incubation was for 60 min at 37°C,after which time the reaction NormalChronic gastritisWith was stopped by the addition of 500 jil of ice cold 0.9% NaCl solution. The atrophyWith tubes were then rapidly centrifuged in a microcentaur centrifuge for 2 min at intestinal metaplasiaSiteAntrum032"1Corpus31863 13,000 g, and the pellet was rapidly washed with ice-cold 0.9% NaCl solution 1One patient with Ménétrier'sdisease. and counted on a Cobra II gamma counter. Characterization and Subtype Determination. In samples where suffi cient tissue was available standard displacement assays were performed using 120 bombesin, gastrin releasing peptide, and neuromedin B as competitors over the concentration range 1 X IO"6 Mto 1 X 10"" M,using the above method, but each point performed in triplicate. The coefficient of variation of triplicates was <10%.

Data Analysis

Total binding was defined as radioligand bound in the absence of competi tors and specific binding as the maximum displaceable binding by the com petitor under investigation, within the defined concentration range. Specific binding by gastrin releasing peptide and neuromedin B was expressed as a percentage of the radioligand displaced by 1 X 10~ft M BBS (% specifically bound). For standard displacement assays the mean (±SEM) of the percentage -10 -9 -8 -7 of specific bound, plotted against the concentration of competitor, enabled log 10[BOMBESIN] (M) calculation of the IC5I) for each peptide. Scatchard analysis was performed Fig. 1. Displacement of 125I-Tyr4-BBS by BBS on human gastric cancer tissue. Mem to determine binding characteristics in terms of binding affinity (K¿)and brane preparations from human gastric cancer tissue were screened for the presence of maximum binding capacity. BBS binding sites. Results shown are the mean (±SEM) of the 13 binding site positive cancers. Inset, a representative Scatchard plot of the displacement data from one assay. Results

Pathological Details 12 of 13 bombesin receptor positive cancer samples. Bombesin bind ing to all gastric cancer samples was to specific, high affinity sites, as General. Tissue was obtainedfrom 16 male and 7 female patients in Fig. 1 [IC5(, = 3.5 (±0.5) HM,Kj = 3.42 (±0.41) DM,and with a median age of 73.5 years (range, 48-87 years). The sites of the maximum number of binding sites = 40.5 (±17.9) pmol/mg protein]. tumors were 12 antral, 7 body, and 4 cardia/gastroesophageal junction. All tumors were adenocarcinoma when examined histologically. The Binding of Bombesin-like Peptides to Binding Sites in Human stage of tumors varied from T,N,>Mnto T3N3M, (Table 1). Cancer. Cancertissue obtainedfrom the above patientswas sub Gastric Cancer jected to routine histology by one histopathologist. The details are Displacement assays performed upon the 12 bombesin binding site demonstrated in Table 1. positive gastric cancers using bombesin, gastrin releasing peptide, and Mucosa. Mucosaobtainedfromthe 23 patientswith gastriccancer neuromedin B revealed that the relative affinities of these three pep- was subjected to routine histopathology by one histopathologist to tides for the binding site were BBS > GRP » NMB (Fig. 2). The ensure that none of the mucosa contained cancer tissue. The informa IC50s, derived from Fig. 2, quantify this difference in affinity: BBS tion is displayed in Table 2. One patient, a 73-year-old male, was found to have Ménétrier'sdiseaseand an early gastric cancer. [3.5 (±0.5)HM],GRP[7.5 (±3.9)HM],andNMB [300.1 (±70.9)HM] (mean ±SEM of the 10 samples in which an IC50was achieved by 1 X 10~6MNMB). Specific Binding of I25l-Tyr4-bombesin to Human Gastric Cancer and Uninvolved Mucosa Discussion Displaceable binding was present in 13 of the cancer samples (57% We have demonstrated high affinity binding sites for a bombesin- like peptide on human gastric cancer tissue and on the mucosa from of cases) and in 1mucosal sample (4% of cases). Sufficient tissue was a patient with Ménétrier'sdiseaseand an early gastric cancer. Other available for binding characterization and subtype determination upon uninvolved mucosa did not demonstrate binding sites for bombesin- like peptides. Table 1 Demographic data and histology of cancer tissue It is known that within the gastric mucosa a subpopulation of cells, TNM staging performed according to the AJC recommendations (Ref. 15); "intestinal" and "diffuse" refer to the Lauren classification of gastric cancer (Ref. 16) the G-cells, express receptors for GRP, demonstrated on cell prepa rations enriched in gastrin cells (17). Our experiments failed to dem Bombesin binding onstrate the presence of these receptors on the antral mucosa exam ined. We assume the explanation is that there are relatively few NumberAge G-cells compared to the large numbers of other epithelial cells within yr]Sex[median (range) in (57-83)9/48/3/2T,NnM(j-TiN2M,I-IVA9/44/2/7Negative1073.5(48-87)7/34/4/2T,N,,M,,-T,N,M,II-1VA5/4"3/0/7our tissue samples. In many gastric cancers, and on the Ménétrier's (male/female)Site (antrum/corpus/cardia)TNM mucosa, there appears to be a relative overexpression of the binding stage(range)StageIntestinal/diffuseDifferentiaiton sites for bombesin-like peptide, enabling detection of these receptors by specific radioligand binding assays. The undetectable levels of (well/moderate/poor)Positive1373 binding sites to bombesin-like peptides on normal mucosa, but their " One cancer could not be classified as either intestinal or diffuse. presence on the hyperproliferative Ménétrier'smucosaand the ma- 5091

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120 targeting of tumor cells. Finally, coupling a BBS-analogue to a radio- O label may be useful in detecting occult métastasesfrom a receptor Z100 positive gastric cancer. Clearly the gastric cancer GRP-binding site o -80 warrants further study.

Acknowledgments I 60 We would like to thank the consultant surgeons and departments of pathol O 40 LU ogy throughout West Yorkshire for providing us with access to their gastrec- CL â„¢¿20 tomy specimens.

References -11 -10 -9 -8 -7 -6 1. Parkin D.M., Laara E., and Muir C. S. Estimates of worldwide frequency of sixteen log, „¿[COMPETITOR](M) major cancers in 1980. Int. J. Cancer, 41: 184-197, 1988. 2. Cancer Research Campaign Factsheets 1.2. Cancer Research Campaign, London. Fig. 2. Standard displacement curves for human gastric cancer tissue. 125I-Tyr4-BBS United Kingdom 1990. displaced by the competitors BBS (X). GRP (•),and NMB (A) over the concentration range 1 X It)-'1 Mto 1 X KT6 M. Results shown are the mean (±SEM) of the 13 gastric 3. Erspamer V.. Falconieri Erspamer G., and Inselvini M. Some pharmacological actions of alytesin and bombesin. J. Pharm. Pharmacol., 22: 275-276, 197(1. cancers for BBS. and 12 of 13 gastric cancers for GRP and NMB. 4. McDonald T. J., Jornvall H., Nilsson G., Vagne M., Bloom S. R., and Mutt V. Characterization of a gastrin releasing peptide from porcine non-antrai gastric tissue. Biochem. Biophys, Res. Commun., 90: 227-233, 1979. lignant tissue of 57% of gastric cancers, suggests that overexpression 5. von Schrenck T., Heinz-Erian P.. Moran T.. Mantey S. A., Gardener J. D.. and Jensen of these binding sites may be an important step in the malignant R. T. in esophagus: evidence for receptor subtypes of bom besin receptors. Am. J. Physiol., 256: G747-G758, 1989. transformation of gastric mucosa. 6. Spindel E. R., Guadi E., Brehm P., Goodman R. H., and Segerson T. P. Cloning and There are several possible mechanisms by which GRP receptors functional characterization of a complementary DNA encoding the murine fibroblast could result in mitogenesis in premalignant gastric mucosa and gastric bombesin/gastrin releasing peptide receptor. Mol. Endocrino!.. 4: 1956-1963. 1990. 7. Sunday M. E., Kaplan L. M., Motoyama E., Chin W. W., and Spindel E. R. Gastrin cancer. The effect may be direct; GRP has known mitogenic properties releasing peptide (mammalian bombesin) gene expression in health and disease. Lab. in Swiss 3T3 fibroblasts and in human small cell lung cancer. It is Invest., 59: 5-24. 1988. present within the gut wall, at highest levels within the stomach, and 8. Wada E., Way J., Shapira H., Kusano K., Lebacq-Verheyden A. M.. Coy D. Jensen R., and Battey J. cDNA cloning, characterization, and brain region- specific expression of the overexpression of receptors may be all that is required to respond a neuromedin-B-preferring bombesin receptor. Neuron, 6: 421-430, 1991. to an already present peptide with mitogenic potential. It has been 9. Fathi Z.. Corjay M. H., Shapira H.. Wada E., Benya R., Jensen R., Viallet J., Sausville demonstrated that in the human pancreatic cancer cell line, MiaPaCa2, E. A., and Battey J. F. BRS-3: a novel bombesin receptor subtype selectively ex pressed in testis and lung carcinoma cells. J. Biol. Chem.. 268: 5979-5984. 1993. BBS causes up-regulation of the EOF receptor by phosphorylating the 10. Tatsuta M., lishi H., Baba M.. Nakaizumi A., Ichii M., and Taniguchi H. Promotion receptor protein (18). This indirect mechanism of action may make the by bombesin of gastric carcinogenesis induced by /V-methyl-/V'-nitro-N-nitroguani- gastric mucosa/cáncer more sensitive to the peptide growth factors dine in Wistar rats. Cancer Res., 49: 5254-5257, 1989. 11. Weber S., Zuckerman J. E.. Bostwick D. G., Bensch K. G., Sikic B. I., and Raffin T. such as EGF and TGFa. The overexpression of TGFa can produce all A. Gastrin releasing peptide is a selective mitogen for small cell lung carcinoma in the histológica! features of Ménétrier'sdisease,as demonstrated by vitro. J. Clin. Invest.. 75: 306-309, 1985. elegant MT-TGFa transgenic mice studies (19). It is also known that 12. Radulovic S., Miller G., and Schally A. V. Inhibition of growth of HT-29 human colon cancer xenografts in nude mice by treatment with bombesin/gastrin releasing peptide many gastric cancers express EGF receptors, and that in certain hu antagonist (RC-3095). Cancer Res., 57.' 6006-6009, 1991. man gastric cancer cell lines, including St42, a mitogenic response is 13. Preston S. R., and Primrose J. N. Gastrin releasing peptide receptor expression on human gastric cancer cell lines. Gut, 34: Suppl., S15. 1993. present to TGFa/EGF (20). BBS also results in release of a wide range 14. Bradford M. M. A rapid and sensitive method for the quantitation of microgram of enteric peptides which may also play a role in regulating gastric quantities of protein utilising the principle of protein-dye binding. Anal. Biochem., mucosal proliferation. 72: 248-254. 1976. 15. AJC (American Joint Committee for Cancer Staging and End Results Reporting). The presence of detectable high affinity binding sites to GRP on Manual for Staging of Cancer. American Joint Committee, 1977. gastric cancer but not on surrounding mucosa may provide new thera 16. Lauren P. The two histological main types of gastric carcinoma. Diffuse and so-called peutic options in the treatment of over 50% of gastric cancers. A intestinal type carcinoma. An attempt at a histoclinical classification. Acta Pathol. Microbiol. Scand.. 64: 31^t9, 1962. number of stable, high affinity BBS/GRP antagonists are currently 17. Vigna S. R., Giraud A. S., Mantyh P. W., Soil A. H., and Walsh J. H. Characterization available as research tools and may be exploited to inhibit the possible of bombesin receptors on canine antral gastrin cells. Peptides, 11: 259-264, 1990. 18. Lee M. T.. Leibow G.. Krebs L. J., and Schally A. V. Bombesin and gastrin releasing mitogenic effects of GRP on gastric cancer. This possibility requires peptide (GRP) induce phosphorylation and up-regulation of epidermal growth factor examination in animal models. In addition, we have previously shown receptor. Proc. Am. Assoc. Cancer Res., 34: 244, 1993. that the BBS/GRP receptor-ligand complex is rapidly internalized in 19. Dempsey P. J., Goldenring J. R., Soroka C. J., Modlin I. M., McClure R. W., Lind C. the human gastric cancer cell line St42/' If this process also occurs in D., Ahlquist D. A., Pittelkow M. R., Lee D. C., Sandgren E. P., Page D. L., and Coffey R. J. Possible role of transforming growth factor a in the pathogenesis of Ménétrier's gastric cancer tisssue then it may be possible to exploit this process by disease: supportive evidence from humans and transgenic mice. Gastroenterology, linking a cellular toxin to a stable BBS-analogue enabling selective 703: 1950-1963, 1992. 20. Durrant L. G., Watson S. A., Hall A., and Morris D. L. Co-stimulation of gastroin testinal tumour cell growth by gastrin, transforming growth factor a and like ' Unpublished data. growth factor-I. Br. J. Cancer, 63: 67-70, 1991.

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Shaun R. Preston, Linda F. Woodhouse, Steven Jones-Blackett, et al.

Cancer Res 1993;53:5090-5092.

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