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[CANCERRESEARCH55,280-287,January15,19951 Characterization of /-releasing Receptors in Human Cancer and Their Relationship to Steroid Expression1

Gabor Halmos, James L. Wittliff, and Andrew V. Schall? Endocrine. Polypeptide and Cancer Institute. Veterans Affairs Medical Center, New Orleans, Louisiana 70146. and Section of Experimental Medicine. Department of Medicine. Tulane University Medical School, New Orleans. Louisiana 70112 (G. H.. A. V. S.]: and Receptor Laboratory, James Graham Brown Cancer Center, University of Louisville. Louisville, Kentucky 40292 Ii. L W.J

ABSTRACT most of our knowledge is derived from studies in experimental animals (5). Bombesin (BN) and its mammalian counterpart, gastrin-releasing pep Breast carcinoma, a disease of the mammary epithelium, is the tide (GRP), are hormonally active which appear to function as autocrine or paracrine growth factors in a variety of cells.As part of a second leading cause of cancer-related deaths among American long-term investigation ofthe relationship of peptide and women (6). In 1993, more than 180,000 new cases of breast carci receptors to breast cancer progression and treatment, we examined the noma were diagnosed in women in the United States alone. Approx binding of [IasI@Tyr4]BN to membranes isolated from 100 human breast imately 48,000 women in the United States will die from this disease carcinomas, Thirty-three of these tumors expressed BN/GRP receptor in 1994 (6). More than 1 million women in America are currently levels of>1O fmol/mg membrane . Two classes of [Tyr@]BN-binding living with stage I, II, or III breast cancer, which indicates the sites were detected using Scatchard analyses of radioligand association magnitude of this health problem. data from hormone displacement curves. The high-affinity binding sites Approximately 30% of women who present with node-negative exhibited a mean dissociation constant (1@d,)of2.1nsi and a mean specific primary breast cancer are at high risk for early recurrence of meta binding capacity (B@1) of 237 fmol/mg membrane protem The low affinity binding sites had a mean dissociation constant (K@)ofO.3 pMand static disease and decreased overall survival (7). Although most breast a mean binding capacity (B,,,,,@)of 5.9 pmol/mg membrane protein. cancer patients with node-positive disease are at high risk for de BN/GRP receptor expression in a breast carcinoma was unrelated to creased disease-free survival and overall survival, 30% appear to be at patient age. When the levels of BN/GRP receptors were compared to the low risk, considering their clinical course. One of the objectives in the content of the sex steroid receptors, a highly significant positive correla application of molecular endocrinology to the problem of breast tion (P < 0.005) was observed between the binding capacities of high cancer is to distinguish these high risk patients from those with low affinity [Tyr@]BN-blndingsites and receptor levels and between risk using clinical laboratory tests (7, 8). the concentrations of low affinity [Tyr4JBN-blndlngsites and progestin The presence of estrogen and progestin receptors in a breast tumor receptor levels (P < 0.05). This represents the first report of these labile, biopsy serves as a predictive indicator of therapeutic response to regulatory In biopsies ofhuman breast carcinomas. Expression of endocrine treatments, such as tamoxifen and medroxyprogesterone specific receptor proteins for BN/GRP, potent mitogens, in a large number of human breast cancers suggests that they may be involved in tumor cell acetate (9, 10). These receptor proteins are also prognostic indices of progression. The approach based on determination of BN/GRP receptors a patient's clinical course (7, 8). Recently, overexpression of EGF might be useful to guide a hormonal therapy with BN/GRP antagonists in receptors in breast cancer (2, 4) has been correlated with decreased some women with breast cancer. disease-free interval and overall survival (3, ii). Thus, these receptors and other cellular expression products appear to be useful as prog INTRODUCTION nostic factors in clinical management of breast cancer. As part of a long-term investigation to define the molecular mech The differentiation and development of the human breast including amsms of endocrine control of breast cancer, we have systematically its function during and are regulated by a variety evaluated human tumor biopsies for hormone receptors. In addition to of including i7f3- and , , the extensive evaluation of sex steroid receptors (8, 12), we detected hormones, and certain peptide hormones, such as , prolactin receptors in human breast cancer (13) although no clinical , and (1). There is increasing evidence that utility of this finding could be demonstrated thus far. We have growth factors, particularly , are involved in likewise characterized EGF receptors in human breast (4), endome development and function of normal and neoplastic breast (2—4). trial (i4), and ovarian carcinomas.4 Specific, high-affinity binding and EGF3 may influence the mitotic state of mammary sites for -releasing hormone and epithelial cells and appear to be involved in ductal development, while were also identified in human mammary tumor cell lines and in biopsy progesterone promotes differentiation of these cells resulting in lob specimens of breast cancer by our group (4, 14) and by others ular development. Prolactin acts synergistically with estrogen and (15—17).Our long-term goal is to exploit the presence of hormone progestin to bring about ductal and lobuloalveolar cell development. binding mechanisms for new therapeutic approaches in breast and Prolactin is also the principal hormone that stimulates lactogenesis. other endocrine-responsive cancers. The exact roles of the other hormones are less well understood, and Recent evidence suggests that tetradecapeptide bombesin, origi nally isolated from amphibia, and its mammalian counterpart gastrin Received 6/10/94; accepted 11/11/94. releasing peptide can function as growth factors, and through an The costsof publicationof this articleweredefrayedin partby thepaymentof page aotocrine or paracrine mechanism it may modulate the growth of charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. some benign and neoplastic tissues (18, 19). BN-like peptides func @ This work was supported by NIH Grant CA 40003 and the Medical Research tion as potent mitogens in vitro and have been implicated as autocrine Services of the Veterans Affairs Medical Center (to A. V. S.) and by a Phi Beta Psi growth factors in the pathogenesis and progression of some human Sorority grant to J. L. W. The contents of this paper are solely the responsibility of the authors and do not necessarily represent the official view of the National Cancer Institute. small cell lung carcinomas (18, 20, 21). Various studies also demon 2 To whom requests for reprints should be addressed, at Veterans Affairs Medical strated that bombesin/GRP may be involved in the function and Center,1601PerdidoStreet,NewOrleans,LA70146. 3 The abbreviations used are: EGF, epidermal growth factor; GRP, gastrin-releasing peptide; BN, bombesin; ER, estrogen receptor; PR, progestin receptor; BNR, [Tyr4J- 4 G. Srkalovic, A. V. Schally, J. L Wittliff, T. G. Day, and E. L Jenison, manuscript bombesin receptor. in preparation. 280

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1995 American Association for Cancer Research. BOMBE5IN/ORP REcEFrORS IN HUMAN BREAST CANCER growth of human breast cancer (22—27).BN-like immunoreactivity centrifugation. All assays were performed in duplicate, and specific binding has been reported in rat mammary tumors (28), in a small proportion capacities and apparent dissociation constants (K,,s) were calculated according of human breast cancer and breast carcinoid specimens (25), in human to the procedure of Scatchard (36) using Lundon One-Site software (Lundon and animal milk (29), and in normal bovine mammary epithelium Software, Inc., Cleveland, OH). Using clinical criteria established by the (i9). It has also been shown that the BN/GRP antagonist RC-3095 National Surgical Adjuvant Breast and Bowel Project (37), tumors containing inhibits growth of estrogen-dependent and -independent MXT mam at least 10 fmol/mg cytosol protein were considered positive for the receptors. mary cancer in mice (30) and MCF-7 MIII human breast cancer in Membrane Preparation. Portions of intact human breast cancer speci nude mice (31). In addition, specific receptors for bombesin/GRP mens (300—400mgeach) were shipped on dry ice from the Hormone Receptor have been demonstrated in human breast tumor cell lines, MDA-MB Laboratory, University of Louisville (where estrogen and progesterone recep 231, MCF-7 MIII, and T47D, but were not detected in MCF-7 and toss were determined), to the Endocrine, Polypeptide and Cancer Institute in BT-20 cell lines or in normal breast epithelial cells (23, 31, 32). New Orleans, where the membrane receptor assays were performed. Prepara Three receptor subtypes associated with the bombesin-like peptides tion of membranes for receptor studies was carried out as described previously have been described and cloned. The cloned receptors have been (4, 38) with some modifications. The samples were thawed and fat debris was distinguished by their relative affinity for binding a series of specific removedusing cotton swabs. Immediatelyafter this, tumorspecimens were ligands. They are classified as: the GRP-preferring subtype (GRP homogenized(1:5,w/v) in 50 mt@iTris-HCIbuffer(pH 7.4)containing5 m@i receptor), found in the intestine from the esophagus to the rectum EDTA-5 mM MgCl2-30 pg/ed bacitracin on ice using an Ultra-Turrax (IKA (19); the -preferring subtype, present in the esophageal Werk-Tekmar, Cincinnati, OH) homogenizer at maximal speed (stroked 3 times for 5 5at 15-s intervals). The homogenate was centrifuged at 500 X g for muscularis mucosa (33); and the bombesin receptor subtype 3, present 10 min at 4°C,and the layer was removed carefully. The supernatant in the testis and lung cancer cell lines (21), the natural ligand of which containing the crude membrane fraction was centrifuged again at 70,000 X g is not yet known. GRP receptor subtypes bind GRP and bombesin for 50 min at 4°Cin a Beckman L8-80 M ultracentrifuge. The pellet was with high affinity and neuromedin B with lower affinity. In contrast, washed once by first resuspendmg it in an ice-cold homogenization buffer and the neuromedin B receptor subtype has a higher affinity for neuro then sedimenting it as before. The final pellet was resuspended in homogem medin B than for GRP or bombesin (34). All three subtypes belong to zation buffer and stored at —86°Cuntilused for the bombesin receptor binding the serpentine receptor superfamily and are coupled through G pro studies. Limited results are available regarding the stability of these receptors teins to effectors including phospholipase C. to freezing since they have been described only recently (35). During our Collectively these observations strongly suggest the need for eval study, bombesin binding was performed immediately after membrane prepa ration and again 3—4weekslater as a control in biopsies with sufficient . uation and characterization of bombesin/GRP receptors in human No significant changes in either K,,s or receptor levels were observed. The breast cancer biopsies to enhance our understanding of the mecha storage of human breast tumor biopsies at —86°Ciswell accepted for retarding nisms involved in these responses. In this study, we report for the first the degradation of steroid and receptors and certain time the presence of specific high-affinity binding sites for [Tyr4]BN (4, 8). Protein concentration was determined by the method of Bradford (39) in human breast cancer specimens. Biopsy specimens from 100 using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA). For human breast cancers were analyzed simultaneously for [Tyr@]BN receptor studies, the membrane fractions were diluted with homogenization binding sites on membrane preparations and estrogen and progester buffer to a protein concentration of 20—50 @W50hi. one receptors in cytosol in order to correlate the levels of expression Radlolodinatlon of [Tyr@]Bombesin and Bombesin Receptor Assay. of these receptors. [Tyr@]BNwas radiolabeled with ‘@‘I-labeledsodiumusing an Enzymobead iodination kit (Bio-Rad, South Richmond, CA) as described (40). Mono-[@I Tyr@JBN was purified by reversed-phase HPLC on a SynChropak RP-8 col MATERIALS AND METhODS umn utilizing a gradient elution with 0.1% trifluoroacetic acid- and acetoni trile-containing eluents. Radioiodination with this method generally yielded Chemicals. [Tyr'@@Bombesinwaspurchased from Bachem (Torrance, CA) 100—200 @Ciof purified [‘1'I-Tyr4]BN. Since the mono- and diiodinated while [‘@I-Tyr'@]bombesinwas prepared in our laboratory using radioisotope [Tyr4]BNs were isolated in pure form, separated from each other and from the 1@1-labeled sodium from Amersham (Arlington Heights, IL). 17@- noniodinated peptide and oxidation products, a theoretical specific activity of [H3jestradiol and [H3]R5020 were purchased from NEN Products/DuPont 1800—2000Ci/mmol was calculated. The adsorption of mono-[@'I-Tyr4)BN to (Wilmington, DL). Other peptides and chemicals were obtained from Sigma different types of surfaces such as borosilicate glass, polystyrene, polypro (St. Louis, MO), Pierce (Rockford, IL), Baxter (Muskegon, MI), Debiopharm pylene, Aquasil-coated glass, Sigma coated glass, and siiconized polypro SA(Lausanne,Switzerland),orAmericanPeptideCompany,Inc.(Sunnyvale, pylene was evaluated. Since essentially no surface adsorption occurred using CA). borosilicate glass tubes, these tubes offered the most favorable conditions. Preparation of Breast Cancer Specimens. Biopsies of human breast Ligand competition experiments were carried out in 12 x 75 mm borosili cancer were submitted for clinical determinations of sex hormone receptors. cute glass tubes in a total volume of 150 p.1binding buffer [25 mMTris-HC1, All analyseswerefirstconductedtomeetthe primaryclinicalrequirementfor 5 mM EDTA, 5 mM MgCl2, 0.25 mr@iphenylmethylsulfonylfluoride, 10 mM patient management; only residual tissue was used for this study. After surgical monothioglycerol, 30 @&g/mlbacitracin, and 0.1% BSA (pH 7.4)] for 60 mm at removal, a histopathological examination of each specimen was performed 24°C.Membranehomogenates were incubated in duplicate or in triplicate with immediately, and a portion of the biopsy was frozen on dry ice and stored at approximately 0.15 [email protected][‘@I-Tyr4Jbombesinas radioligand and with increasing —86°Cuntilthe analyses of steroid and peptide hormone receptors. Patient age concentrations (10_12_10_6 M) of unlabeled [Tyr4jbombesin or structurally ranged from 29 to 94 years. unrelated peptides as competitors. The reaction was terminated by adding 250 Determination of Estrogen and Progestin Receptors. Estrogen and pro @ii ice-cold binding buffer to the tubes, and the bound ligand was separated gestin receptors were determined by radioligand binding using a titration assay from free ligand by centrifugation at 4500 X g for 10 mm at 4°C.Thepellet (35). Briefly, cytosol was preparedas described using 10 mMTris-HC1buffer was washed twice with 500 gil ice-cold buffer, and the radioactivity in the (jH 7.4), containing1.5 mMEDTA, 10 mMmonothioglycerol,10%glycerol, pellet of each tube was counted in a gamma counter (Micromedic Systems, and 10 mM sodium molybdate. Protein concentrations of the cytosol were Inc., Huntsville, AL). Some experiments were performed with membrane adjusted to 2 mg/ml and incubated with either 17P-[3Hlestradiolor [3H]R5020 protein concentrations ranging from 10 to 100 @gltubeinorder to determine at 6 concentrations ranging from 0.2 to 4 n@iat4°Cfor16—18h.Nonspecific the minimal amount of protein required to assess specific binding at a antis binding was assessed in the presence of a 200-fold excess of either unlabeled factory level. diethylstilbestrol or R5020 for estrogen and progestin receptors, respectively. Mathematical Analysis of the Binding Data. Estimates of specific ligand Unbound hormone was separated from bound labeled steroid by incubation binding capacities and affinities were calculated by the Ligand-PC computer. with 0.3 ml of a dextran-coated charcoal suspension for 15 mm followed by ized curve-fitting program of Munson and Rodbard (41) as modified by 281

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1995 American Association for Cancer Research. BOMBESIN/GRPRECEPTOR5INHUMANBREASTCANCER McPherson (42) and the Lundon-2 competition data analysis program (Lundon 1000 , I II Software). To determine the types of receptor binding, dissociation constants SIIS•S.S (K@,s) and the maximal binding capacity of receptors (B@@j, bombesin binding 900 data were also analyzed by the Scatchard method (36). Correlation coefficients (r) were calculated by a computer program. P <0.05 was accepted as a statistically significant difference. BOO

C

4-, 700 RESULTS ‘-I Expression of Sex Hormone Receptors in Tumor Biopsies. To 0@0. o.@w 600 ensure that the population of human breast carcinomas utilized in this w U) 5 investigation of bombesin receptor expression was representative of S55SSSSSS. 500 both potentially endocrine-responsive and -unresponsive tumors, sex hormone receptors were examined in the cytosol preparations. Exam ination of estrogen receptor levels in biopsies as a function of patient 400 age is presented in Fig. 1. Utilizing 50 years of age or younger for premenopausal status, the median estrogen receptor level was 45 300 fmol/mg cytosol protein (n = 29). The median estrogen receptor level S••S was 98 fmol/mg for biopsy specimens from postmenopausal patients 200 (n 71). S S Fig. 2 examines the relationship of patient age and progestin re 100 S ceptor levels in breast carcinomas. The median progestin receptor .%S. S @ level was 17 fmol/mg for premenopausal patients (n = 29), while that ‘ ..l.:s!.i@ds5@• ..- for postmenopausal patients (n = 71) was 44 fmol/mg. 20 30 40 50 60 70 80 90 100 In general, progestin receptor level rises as a function of an increase in estrogen receptor level (e.g., 12). When this relationship was ACE (yr) examined in the tumor population studied (Fig. 3), the equation of the Fig. 2. Relationship of patient age and progestin receptor levels in human breast line expressing the correlation was carcinomas. Progestin receptor levels were determined as described in “Materialsand Methods―using cytosols from 100 biopsies of human breast cancer. Specific binding y = 0.97x + 7.28 (1) capacity of each preparation is expressed as fmol/mg cytosol protein. The biopsy, from a 94-year-old patient, containing 3654 fmol/mg cytosol protein does not appear on the (r 0.64). Utilizing a value of 10 fmol/mg for estrogen receptor figure. negativity, the median progestin receptor level was 5 fmol/mg

(n 36); estrogen receptor-positive tumors had a median progestin

@@ 1800 — receptor level of 145 fmol/mg (n 64). Utilizing a value of 10 fmol/mg for progestin receptor negativity, the median estrogen recep tor level was 10 fmol/mg (n = 47); progestin receptor-positive tumors 1600 exhibited a median value of 172 fmol/mg estrogen receptors (n = 53). Characterization of [Tyr'@1BNReceptorsin Biopsies. Of the 100 tumor preparations examined, 27 contained sufficient membranes of a:: high integrity resulting in Scatchard plots satisfactory for detailed

@ .@ 1200 analyses of specific [‘@I-Tyr4]BNbinding. In six additional cases, limited quantities of tumor membrane fractions indicated the presence Ui of [Tyr4]BN receptors. Using a single saturating dose of ligand, 67 @ Ui 1000 biopsies did not exhibit [Tyr4]BN receptors regardless of the proce dure applied. Using a computerized curve-fitting program, typical @ Ui 800 displacement curves were obtained. The nonlinear curve fitting and the Scatchard plot analyses suggested that the labeled [Tyr4]BN was S associated with two classes of receptor sites (Fig. 4). In 27 tumors S analyzed by complete displacement, the high-affinity binding sites WE I. exhibited a mean dissociation constant (KdJ) of 2.1 ±1.8 (SD) fiM -@-, 400 S S S (range, 0.1—7.8fiM)and a mean maximal binding capacity (B,,@1) of 237 ±222 fmol/mg membrane protein (range, 12—1050fmol/mg 55 5 protein). The low affinity sites bound bombesin with a mean disso @ 200 555 : @@ .51 @S ciation constant (Kd2) of 0.3 ±0.2 p.M (range, 0.01—1.3 @.tM)anda ._@t@@_1p@S - S mean maximal binding capacity (Bmax2)of 5.9 ±5.5 pmol/mg mem 0— 20 30 40 50 60 70 80 90 100 brane protein (range, 0.6—25 pmol/mg protein). In addition, more than 24% of BNR-positive samples displayed relatively elevated values (>100 fmol/mg membrane protein) of high-affinity [Tyr']BN-binding ACE (yr) sites. From our experience with the expression of other peptide Fig. 1. Relationship of patient age and estrogen receptor levels in human breast hormone receptors (4, 8, 14), levels greater than 100 fmol/mg carcinomas. Estrogen receptor levels were determined as described in “Materialsand Methods―using cytosols from 100 biopsies of human breast cancer. The specific binding membrane protein are “elevated―relativeto those observed in capacity of each preparation is expressed as fmol/mg cytosol protein. nonmalignant breast tissue. 282

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1000 [Tyr―]bombesin,GRP(14—27), and neuromedin B (Fig. 5). While the S binding of [‘@I-Tyr4]BNwas completely displaced by increasing 900 concentrations (10 12_10_6 M) of nonlabeled [Tyr4]BN and S GRP(14—27@,none of the structurally or functionally unrelated pep Boo tides tested inhibited binding of labeled [Tyr―]BN at 10_6 M. [Tyr4]BN and GRP(14—27)exhibited equally high affinity for the C receptor subtype while neuromedin B was less effective by two orders 700 5 5 of magnitude in displacing [1@I-Tyr4]BN binding (Fig. 5). Displace S S 0-0. ment by unlabeled [Tyr4]BN (Fig. 5) gave an apparent dissociation w 000 constant virtually identical to that obtained for the high-affinity re I captor determined by radioligand binding (Fig. 4). 4-' SOC Influence of Membrane Protein Concentration on [1251 I— SS Tyr―]BNBinding in Human Breast Cancer. A common error in (flat WE 400• S receptor measurements arises from the instability of receptor binding at low protein concentrations. This point was assessed in two exper @ 3005 S S iments in which human breast cancer membrane fractions prepared S I. S in homogenization buffer were serially diluted in the same buffer 200 S •@ and assayed for [‘@I-Tyr4]BN binding. This work indicates that S S S accurate results can be obtained over a wide range (20—100p@g/tube) @ 100 S of membrane protein concentrations. For most assays, protein con @@ S centration in the incubates was maintained at approximately 20—50 0 PV@@LIh:1@4!@. @‘# I I @g/50p1. 0 200 400 600 800 1000 Correlation of Bombesin Receptors with Expression of Estro gen and Progestin Receptors. Table 1 and Figs. 6 and 7 show the ESTROGEN RECEPTOR (fmol/mci cytosol protein) Fig.3. Interrelationshipofestrogenandprogestinreceptorexpressioninhumanbreast caranoinas. The results of analyses of two carcinoma biopsies do not appear on the figure; onebiopsycontained1654tool/mgproteinestrogenreceptorsand3654tool/mgprotein x 100 pro_n receptors,whilethe secondbiopsycontained1277fmol/mgproteinestrogen 0 receptorsand512tool/mg proteinprogestinreceptors. E 90 0 80 —:100 0.05 x U Kd1.1J3 M @ 0.04 I@@z$@S@t@sL/@S 70 E I― @ I― W..03 z @ 0.03 [email protected]/_I p―I. 60 0 I'. a z 50 0 ,@ >@ z I- 40

In c',1 30 ,* 0 500 1000 1500 2000 20 ;40 BOUND(pM) z 10 0 @‘20 0 —12—11—10—9—8—7—6—5 log COMPETITOR (M) 0 Fig. 5. Ligand-binding specificity of [Tyr4]bombesin binding. Competition for binding —11—10 —9 —8 —7 —6 —5 —4 of labeled [Tyr4jbombesin to human breast cancer membranes was determined in the presence of increasing concentrations of epidermal growth factor (V), human growth hormone-releasing hormone (V), somatostatin (V), [D-Trp6J-luteinizing hormone-releas log [Tyr4]—BN(M) ing hormone (V), [Tyr@]bombesin(0), gastrin-releasing peptide(14—27)(5), and neuro Fig.4.Representativeexampleofligandcompetitionassayof[Tyr4jbombesinbinding. medin B (Y). One hundred %-specific binding is defmed as the difference between A constantamountof [‘@l-Tyr4@tumbesinwasincubatedwith increasingamountsof binding in the absence and in the presence of lO_6 M[Tyr4JBN.Point, mean of at least unlabeled [Tyr@]bombesinusinga membrane preparation of human breast cancer. Specific three experiments, each performed in duplicate. binding was determined at each concentration as described in “Materialsand Methods.― Eachpoint representsthe mean of triplicatedeterminations.Inset,Scatchardplot of specific [tasI@Tyr'@Jbombesin@bindingdataanalyzed by a two-site model. TabletoBombesin 1 Distribution of bombesin receptors in human breast carcinomas sex hormone receptor statusaccording statusER@PR@―hormone receptor Specificity of Binding. The specificity of bombesin/GRP binding ER@PRERPR@BNR@―21receptorsSex ERPR to membranesof humanbreastcancer specimens was demonstrated 63BNR29 3 by competitive binding experiments using several peptides such as 28 82 epidermalgrowthfactor,humangrowthhormone-releasinghormone, a ER and PR positivity, 10 fmol/mg cytosol protein. somatostatin, [D-Trp6] luteinizing hormone-releasing hormone, b BNR positivity, >10 fmol/mg membrane protein. 283

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1100 , lished between concentrations of low affinity [Tyr4@BN-bindingsites S and PR-binding capacities (Table 2), there was no correlation between 1000 - the concentrations of high-affinity binding sites of [Tyr@1BNand PR levels (Table 2). Fig. 6 illustrates the relationship between estrogen —@ 900 receptor expression in 100 tumor biopsies and BN/GRP receptor C content. Although most of the tumors did not express bombesin U, @ 800• receptors, it is suggested that for those that do, the binding capacity o@. increases as a function of estrogen receptor activity. The relationship I— S 0-Up 700 5 UJ@ 00 @WL@ 600 700 —I E ZU, cnE Ui 600 ma@@E 400 S 5 C @ S a, 0 300 5 500

‘4—E S S-.- S 200 • S o-q, 00 400 @ S 5 S 100 5 5 S S Za, @ 0 I 15 nE @30O S 400 800 1200 1600 Ui mol S @ ESTROGEN RECEPTOR 0 S (fmol/mg cytosol protein) 200 E S ‘4- S @ Fig. 6. Relationship of estrogen receptor expression and bombesin receptor levels in S 100 human breast carcinomas. Bombesin receptors were determined on membrane prep arations remaining after extraction of cytosol using procedures described in “Materials 100 5 5 @S S and Methods.―Only high-affinity bombesin receptor levels are plotted. S

0 200 400 600 800 1000 distribution of tumors containing [Tyr4JBN receptors as a function of estrogen and progesterone receptor status in 100 human breast cancer PROGESTIN RECEPTOR specimens examined. Tumor tissue samples containing BNR greater (fmol/mg cytosol protein) than 10 fmol/mg membrane protein were arbitrarily designated as Fig. 7. Relationship of progestin receptor expression and bombesin receptor levels in positive while ER and PR values 10 fmol/mg cytosol protein were 100 human breast carcinomas. Bombcsin receptors were determined on membrane prep considered positive according to clinical response criteria established arations remaining slier extraction of cytosol using procedures described in “Materials by the National Surgical Adjuvant Breast Project (37). Membrane andMethods.―Theresultsof analysesontwocarcinomabiopsiesdonot appearon the figure; one biopsy contained 261 finol/mg protein progestin receptors and 1050 fnsol/mg preparations from 33 of 100 cases (33%) exhibited receptors for protein bombesin receptors while the other biopsy contained 3654 fmol/mg protein [Tyr4}BN. As shown in Table 1, ER was detected in 82% (27 of 33) progestin receptors and 724 tool/mg protein bombesin receptors@Only high-affinity and PR in 73% (24 of 33) of the tumors containing bombesin recep bombesinreceptorlevelsareplotted. tors with the characteristics of positivity defined above. Six of the 36 ER-negative tumors (17%) contained specific [Tyr4JBN receptors. Table 2 RelationshipandPR between the binding capacities of [Tyr@JBNand those of ER Twenty-eight tumors did not contain significant levels of BNR, ER, or receptors in humanbreast cancerspecimensReceptor[Tyr@]BN}ligh PR using the criteria described while 21 of 100 (21%) breast cancer specimens expressed all 3 receptors. Previous reports have shown that expression of the sex steroid LowType hormone receptors was positively associated (2, 4). In this study, affinityERof receptor ER PR affinity when all sex steroid receptor values were considered, a statistically 0.385n―=SO0378― 0.671 significant positive correlation was found between binding capacities n=23P<0.005 n=27 of estrogen and progestin receptors (r = 0.641; P < 0.001; n = 100) NSPR P<0.005 (Fig. 3). When only receptor values with at least 10 fmol/mg cytosol 0.541n=500.578 0.311 protein were considered, a statistically significant correlation n=18P<0.005 n=21 (r 0.578; P < 0.005; n 50) (Table 2) was also found, indicating P<0.05[Tyr4JBN NS that ER and PR are not independent variables. To determine whether (high affinity)0.135n=27 0.671 0.311 bombesin/GRP receptor concentrations were correlated with the cx n=27P<0.005 n=21 pression of either ER or PR, the data were analyzed by a computer NS[Tyr4JBN NS correlation test. A positive correlation, highly significant statistically, (low affinity)0.135n=23 0.385 0.541 was shown between binding capacities of high-affinity [Tyr4]BN n=27NS n=18 NSa P<0.05 binding sites (receptors) and ER concentrations (Table 2). No corre Correlation coefficient (r). lation was observed between [Tyr4]BN binding to low affinity sites b n, number of human breast cancer specimens exhibiting specific binding to both and ER levels (Table 2). Although a positive correlation was estab receptors; NS, statistically insignificant correlation. 284

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ageHumanTable 3 (‘25l-Tyi@'jbombesin-bindingasa function ofpatient [Tyr4]BN and the Scatchard plots of these data indicate two classes of breast cancer specimens were assayed for specific [‘@I-Tyr41BNbindingas binding sites, one with high affinity (mean KdJ = 2.1 nM) and low deacn@bedin“MaterialsandMethods.―Tumorsamples with 10 fmol or more of [@l TyT@)BNboundspecifically/mgBNRpositive.Age of membrane protein were designated arbitrarily as binding capacity (mean Bm@i 237 fmol/mg membrane protein) and the other with low affinity (mean Kd2 = 0.3 @.LM)andhigh binding Positive<29(yr) Positive/total % capacity (mean Bm@2 5.9 pmol/mg membrane protein). The spec 030—39 0/1 ificity of the receptors for [Tyr'@]BNbinding was studied using com 040-49 0/3 petitive ligand-binding experiments. These receptor proteins recog 4350—59 9/21 nized bombesin and GRP(14—27) peptides in a specific manner with 2060—69 4120 3570—79 6/17 high affinity as ascertained by evaluation of a variety of unlabeled 3880-89 8/21 compounds. Since the affinity of [Tyr4]BN and GRP(14—27) was 36>90 5/14 100-fold higher than that of neuromedin B, we concluded that human 1/3 33 breast carcinomas contain primarily the BN/GRP receptor subtype. In addition, these data suggest that high-affinity binding represents as between BN/GRP receptors and progestin-receptor expression is pre sociation with bombesin/GRP receptors while low affinity binding sented in Fig. 7. These data confirm the analyses shown in Table 2 represents association with neuromedin B receptors. Further investi that shows there was no correlation between the presence of high gation using different ligands will distinguish the receptor subtypes. In affinity binding sites for [Tyr4JBN and PR levels. In addition, no contrast to human breast cancer cell lines, MDA-MB-231, MCF-7 correlationwasobservedbetweenbindingcapacitiesof eitherhighor MIII, and T-47D, in which a single class of high-affinity BN/GRP low affinity [Tyr4JBN-binding sites (Table 2). Finally, no statistically binding sites were detected (23, 31), both high and low affinity significant correlation was found between the dissociation constants binding sites were detected. All human breast cancer specimens, in (Kd, IBIdK@) of the two classes of [Tyr'@]BN-bindingsitesin human our experience, demonstrated both high and low affinity binding sites, breast cancer specimens examined (r = 0.115; n = 27). but the significance of these two types of sites is unclear at present. Relationship of Patient Age and [‘251-Tyr4]BNBinding. The Thirty-three of 100 human breast carcinomas expressed [Tyr4]BN menopausal status of a patient is related to age. Most studies assume receptor levels of >10 fmol/mg membrane protein. The relatively wide that 50 years of age is a useful time to evaluate marker expression as variation in the dissociation constants and the maximal binding ca a function of premenopausal and postmenopausal status. There was no pacity of BN/GRP receptors is not clear at present. The presence of significant correlation of BNR levels with age of patients either when BN/GRP receptors of different affinity subtypes suggests that poly high-affinity [Tyr―]BN-binding sites were considered (r = 0.291; morphism may be related to biological response. Usually, the receptor n = 27) or when low affinity [Tyr―]BN-bindingsites were evaluated isoform exhibiting the higher affmity has been correlated with clinical (r 0.211; n = 27). As shown in Fig. 8 and Table 3, the presence of response to therapy as in the cases of estrogen and progestin receptors BNR in breast carcinomas was not related to patient age, with the (7—9,12) in breast cancer. exception that specific [Tyr'@]BN-binding was not detected in biopsies It is interesting to note that the expression of BN/GRP receptors from patients under 40 years of age. However, only 4 specimens were appears to be related to clonal origin. Some human breast tumor cell examined.

DISCUSSION 1100 S Bombesin, GRP, and related peptides influence the release of 1000 gastrointestinal hormones, stimulate gastric and pancreatic , and gastrointestinal motility (19—21,27,29, 32). Bombesin-like im 900 munoactivity has been demonstrated in the , gastrointestinal C a, tract, lungs, and mammary glands of various animals (19, 22). Bom 800 besin and gastrin-releasing peptide also appear to function as auto @Q. S crine or paracrine growth factors in a variety of cells (18, 19—23,25, 700 S Ui c 26). These studies indicate that bombesin/GRPs are involved in the 00 regulation of cell proliferation and differentiation. Our understanding [email protected] @- 600 of the biological effects of bombesin and GRP suggests that these E peptides utilize membrane-bound receptors as part of their mechanism @5E 500 LU of signal transduction (20, 21, 24, 32). S 400 S As part of a long-term investigation of the relationship of peptide S @ 0 S and steroid hormone receptors to human breast cancer progression and S 300 S treatment, we characterized [Tyr4JBN receptors in biopsy samples of E ‘4- S breast carcinomas. Several studies by our group (27, 31, 43, 44) using S @ bombesin antagonists demonstrated the presence of bombesin/GRP 200 S receptors in human breast, pancreatic, and gastric cancer cells S S 100 S S grown in tissue culture. Giacchetti et a!. (23) also described BN/GRP S SS receptors in certain breast cancer cell lines that were absent in normal 0 breast epitheitun. To our knowledge, this study is the first to identify 30 40 50 80 70 80 90 100 and characterize these receptor proteins in biopsies of human breast carcinomas. ACE (yr) Examination of the binding of [‘@I-Tyr4]BNwas performed using Fig. 8. Relationship of patient age and bombesin receptor expression in human breast membranes isolated from 100 human breast carcinomas. The analysis carcinomas. Bombesin receptor levels were determined by ligand-binding assays as described in “MaterialsandMethods―usingmembrane preparations from breast tumor of complete displacement curves of [@I-Tyr―]BN by unlabeled biopsies. Specific binding capacity is expressed as fmol/mg membrane protein. 285

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1995 American Association for Cancer Research. BOMBESIN/GRP RECEPTORS IN HUMAN BREAST CANCER lines, such as MDA-MB-231 and T-47D, were reported to contain stimulus for the development of BN/GRP antagonists with therapeutic these proteins; while in other lines, such as MCF-7 and BT-20, and in potential. It is also possible that, on the basis of such receptor studies, normal breast epithelial cells, the BN receptors were stated to be highly active bombesin/GRP antagonists could be used for the treat absent (23, 27, 31). However, Patel and Schrey (24) found BN/GRP ment of selected breast cancers in women. binding sites in MCF-7 cells and showed that bombesin stimulated inositol phospholipid and Ca2@ mobilization but did not ACKNOWLEDGMENTS stimulate proliferation. MCF-7 MIII subline isolated from MCF-7 tumors proliferating in nude ovariectomized mice also showed high We are grateful to Dr. Slobodan Miseljic, Katalin Halmos, and Dr. Balazs affinity bombesin receptors (31). Szoke for their valuable help. The duration of remission of breast cancer produced by treatment with antiestrogens and endocrine ablation (ovariectomy) is varied, and REFERENCES many hormone-responsive tumors become unresponsive (7, 10). The presence of both estrogen and progestin receptors in a breast cancer 1. Keller-Wood, M., and Bland, K. I. Breast in normal, lactating, and diseased states. In: K. I. Bland, and E. M. Copeland, III (eds.), The Breast: Compre biopsy indicates a 75—80%likelihood that the patient will respond to hensive Management of Benign and Malignant Diseases, Ch. 3, pp. 36—45. Phila endocrine manipulations (7, 8, 12). Other patients, usually those with delphia: W. B. Saunders Co., 1991. receptor-negative tumors, do not benefit from antihormone therapy. 2. 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Gabor Halmos, James L. Wittliff and Andrew V. Schally

Cancer Res 1995;55:280-287.

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