Ovarian Cancers Express and Process Progastrin1

[CANCER RESEARCH 53, 1823-1828. April 15. 1993] Ovarian Cancers Express and Process Progastrin1

Wouter W. van Solinge, Lars 0dum, and Jens F. Rehfeld2

University Department of Clinical Biochemistry, Rigshospitalel, DK-2IOO Copenhagen. Denmark

ABSTRACT also measured other peptide systems (CCK,3 somatostatin, and Sub stance P) in order to assess the selectivity of the hormone expression. Gastrin synthesis in ovarian tumors has been described in a few isolated cases associated with the Zollinger-Ellison syndrome. Consequently, ova rian gastrin synthesis has been considered exceptional. In order to eval NOMENCLATURE uate whether expression of gastrin in ovarian tumors indeed is rare, we Human preprogastrin comprises 101 amino acid residues (19). It examined the expression and processing of progastrin in 16 malignant and 5 benign ovarian tumors and 4 normal postinennpaiisal ovarÃa.Using a consist of a signal peptide of 21 amino acid residues, a spacer se library of sequence specific radioimmunoassays, cleavage by processing- quence, the sequence containing the major bioactive forms of gastrin like enzymes, and gel chromatography, we found that one-half of the [i.e., gastrin-34 (pGlu 38-Phe 71), and gastrin-17 (pGlu 55-Phe 71)], malignant tumors expressed significant concentrations of amidated gas- and a COOH-terminal-flanking peptide (Fig. 1). In this study the trins [6.7 Â± 2.7 (SEM) pmol/g; range, 1.4-20.0 pmol/g, n = 7]. The following categories of preprogastrin products are distinguished: cat concentrations of glycine-extended gastrins and progastrins were low (0.25 egory I, amidated (bioactive) gastrins, in which the COOH-terminal Â±0.03 and 1.4 Â±0.4 pmol/g, respectively) but higher than in controls and phenylalanine (position 71) residue is amidated; category II, glycine- benign tumors. Chromatography showed that the majority of the bioac- extended gastrins, defined as gastrin molecules processed to Trp- tive gastrins was unsulfated gastrin-17. The other half of the malignant Met-Asp-Phe-G/y at the COOH terminus. These molecules are the tumors expressed glycine-extended gastrins and progastrins (0.2 Â±0.03 and 0.6 Â±0.1 pmol/g; n - 9), but the amidation of the peptides was immediate precursors of the bioactive gastrins; the glycine residue in impaired (0.1 : 0.03 pmol/g). Low concentrations of glycine-extended position 72 being the amide donor (20); category III, progastrins, gastrins and progastrins were detected in the normal ovarian tissues (0.2 which are defined as products extended beyond glycine (position 72) v 0.05 pmol/g tissue and 0.2 Â±0.06 pmol/g, respectively, n = 4) and in the at the COOH terminus; category IV, processing fragments, defined as benign tumors (0.1 Â±0.02 pmol/g and 0.5 Â±0.03 pmol/g; n = 5). Amidated products between monobasic and/or dibasic cleavage sites, devoid of gastrins were undetectable, except in low amounts in a single benign the carboxyamidated COOH terminus. Fragments are denoted by tumor (0.2 pmol/g tissue). The results show that postmenopausal ovaria numbers, indicating the position of the first and last amino acid. Each and neoplasia- ovarian tissues express the gastrin gene at peptide level. of categories I-III contains a number of peptides of different chain The synthesis and processing of progastrin increase considerably in ma length, with or without sulfation of tyrosine-66. lignant tumors.

MATERIALS AND METHODS INTRODUCTION Progastrin matures to bioactive peptides by proteolysis at mono- Tissue. Ovarian tumors were removed at surgery and immediately im mersed in liquid nitrogen. Samples were stored at -80Â°C until extraction. and dibasic cleavage sites and by amino acid derivatizations (sulfation, phosphorylation, and amidation). Of these, phenylalanine car- Tumors were histologically verified and classified (21 ) as shown in Table 1. All patients were postmenopausal. Histologically verified nonneoplastic ovarian boxyamidation is necessary for bioactivity [Fig. 1 (1)]. Bioactive tissues were obtained from postmenopausal patients undergoing hysterectomy. gastrins stimulate gastric acid secretion and fundic mucosal growth (2, The use of human tissues was approved by the local ethics committee. 3). Accordingly, gastrin producing tumors (gastrinomas) lead to re Extraction. While frozen, tissues were minced and immersed directly in current peptic ulcÃ©rationand hyperplasia of the fundic mucosa, i.e., boiling water (pH 6.6), 6 ml/g tissue. The tissues were boiled for 20 min, after the Zollinger-Ellison syndrome (4, 5). which tissues were homogenized and centrifuged at 10,000 x g for 30 min. By far most gastrinomas are located in pancreas or duodenum (6). After dÃ©cantationof the supernatants, the pellets were reextracted in 0.5 M During the last decades ten isolated cases of the Zollinger-Ellison acetic acid for 20 min. homogenized, and centrifuged at 10,000 x g. Super natants were stored at -20Â°C until assay. syndrome due to gastrin secreting ovarian tumors have been reported (7-16). Although ovarian gastrin synthesis in amounts sufficient to Radioimmunoassays. In order to measure all biologically active forms of develop a fulminant Zollinger-Ellison syndrome apparently is ex gastrin and CCK as well as their precursors, a library of sequence specific radioimmunoassays was used. The epitopes of the different antisera used in tremely rare, neuroendocrine cells have been demonstrated as a reg these radioimmunoassays are shown in Fig. 1. Bioactive (carboxy-amidated) ular feature of mucinous ovarian cystadenocarcinomas. Some of these gastrin and CCK peptides were measured using antisera 2604, 2605, and 2609 cells contained gastrin immunoreactivity (17, 18). This observation which all are directed against the common a-carboxyamidated COOH termi prompted us to investigate whether the synthesis of gastrin in ovarian nus of gastrin and CCK. Antiserum 2604 binds all bioactive gastrins (sulfated tumors is a sporadic or a general phenomenon. as well as nonsulfated) with equimolar potency, whereas the cross-reactivity We have now measured the expression and processing of progastrin with CCK is negligible (22). Antiserum 2605 has characteristics identical to in ovarian tumors and normal postmenopausal ovaria using a library those of antiserum 2604, except that it measures only nonsulfated gastrins (22). of sequence specific radioimmunoassays for human progastrin. We Antiserum 2609 measures all biologically active forms of gastrin and CCK, sulfated as well as nonsulfated (22). Glycine-extended gastrin and CCK was measured using antiserum 3208, which is directed against the glycine-extended Received 9/24/92; accepted 2/1/93. COOH-terminal sequences of gastrin and CCK (23). In addition antiserum The costs of publication of this article were defrayed in part by the payment of page 5284 was used. It binds only glycine-extended nonsulfated gastrins (23). charges. This article must therefore be hereby marked advertisement in accordance with Regarding progastrin. proCCK, and processing fragments, antisera 2145 and 18 U.S.C. Section 1734 solely to indicate this fact. 1The study was supported by grants from the Danish Medical Research Council; the 8017 are specific for the NH2-terminal sequence of human gastrin-34 (24) and Danish Cancer Union; and the Alfred Benzon, Carlsberg, Gangsted, NOVO, and Vissing the NH2-terminal sequence of human gastrin-17 (25), respectively. They bind Foundations. 2 To whom requests for reprints should be addressed, at Department of Clinical Bio chemistry (KK-3011). Rigshospitalet. DK-2100 Copenhagen, Denmark. 3 The abbreviation used is: CCK, cholecystokinin. 1823

SR RR KK Y QRR TT T -21 1 5 19 36 37 53 54 66 727374 80 Fig. 1. Structure of human preprogastrin. A. structure and mono- and dibasic processing sites of human preprogastrin. Numbers, characteristic amino acids. Serine I is the first amino acid of progastrin. The glycine residue at position 72 func tions as amide donor in the amidation reaction. Sul- fation can occur at the tyrosine residue in position I h JÂ»yj-Givi I Ab*D 52843208 66. Gastrin-34 is constituted of amino acids Gln-38/ Phe-71 and gastrin-17 of amino acids Gln-55/Phe- Ab. S8235 Ab. 2145 Ab. 8017 71. fi, localization of epitopes in the glycine ex tended gastrins: C localization of epitopes in the carboxyamidated gastrins. For antibody (Ab. ) char acteristics see text.

2604 I (- H, Ab 2605 2609 Ab. 88235 Ab. 2145 Ab. 8017

gastrin-34 and gastrin-17, as well as their COOH-terminally extended precur Table I Concentrations of amidated gastrins and their precursors in extracts of sors. In order to measure NHi-terminal extended precursors as well, tissue ovarian tumors and in nonneoplaslic ovarian tissue tpmal/R tissue, wet weight) extracts were incubated with equal volumes of trypsin (1 mg/ml in 0.05 M Glycine- sodium phosphate, pH 7.5) at 20Â°Cfor 30 min. The enzymatic reaction was Amidated extended Tumor FIGO" gastrins gastrins Progastrins terminated by boiling for 30 min. By tryptic cleavage the NH2 termini of gastrin-34 and gastrin-17 will be exposed for binding to antisera 2145 and MalignantSerous 8017. respectively. Consequently progastrin and its processing products are cystadenocarcinomaI2345678uIO1112Nondifferentiated measured after tryptic cleavage (26). Another way of measuring COOH-ter minally extended precursors is by using antiserum 3208 and/or 5284 after sequential cleavage with trypsin (see above) and carboxypeptidase B (100 ul/ml sample). Both CCK and gastrin precursors are then bound to antisera 3208 and only unsulfated gastrins are bound to antiserum 5284 (23). Recently, we developed a radioimmunoassay directed against the NH2-terminal sequence of human gastrin-52 (progastrin fragment 19-35). The antiserum (antiserum 88235) cross-reacts with reduced sensitivity with fragments unprocessed at 1<0.12.320.01.70.10.30.30.30.10.20.20.20.10.30.10.40.20.40.20.21.30.80.60.60.60.50.80.60.70.21.32.33.10.90.5 arginine-19. Somatostatin was measured using antiserum R-433, which recog nizes somatostatin-14 and prosomatostatin (27). Substance P was measured carcinoma131415nimHIUIunIVinHIIVIII1CIIIHIIVIII12.95.91.42.70.20.1<0.1<0.10.1<0. with an antiserum directed against the carboxyamidated COOH terminus of substance P. For measurement in tissue extracts, the detection limit of all assays is <0.l pmol/g (wet weight). Serous cystadenocarcinoma of Chromatography. One ml of tumor extract (Table 1, tumors 1 and 13) was borderline malignancy applied to Sephadex G-50 superfine columns ( 10 x 1000 mm) and eluted with 16 0.2 0.2 0.5 0.125 MNHjHCO, (pH 8.2) at 4Â°Cwith a flow rate of 4 ml/h in fractions of 1.0 ml. Void and total volumes of the columns were determined with I25I- Benign Serous cystadenoma labeled albumin and "NaCI. Columns were calibrated with human gastrin-17, 17 0.1 0.4 human gastrin-34. fragments 38-54 and 19-35 of human progastrin. human 18 0.2 0.1 0.5 ,11111,i!mucosa extract. CCK-33. and CCK-8. Tumor extracts were applied to 19 0.2 0.5 Mucinous cystadenoma the column and fractions were assayed with the antisera (see above). In 20 0.6 addition fractions were treated with trypsin and carboxypeptidase B (see above) and assayed with appropriate antisera. Alternatively, extracts were Follicular cyst treated with enzymes prior to application to the column, and the fractions were 21 0.2 0.5 Controls'" 0.2 Â±0.05 0.2 Â±0.06 subsequently assayed. " Classification according to International Federation of Gynecology and Obstetrics (FIGO) (21). *Mean Â±SEM (n = 4). RESULTS As shown in Table 1,12 tumors were classified as serous cystadenocarcinomas, 3 were classified as cystadenomas, 3 carcinomas were cine-extended gastrins and progastrins were 0.1 Â±0.02 pmol/g and epithelial but nondifferentiated (tumors 13, 14, and 15), 1 tumor was 0.5 Â±0.03 pmol/g (n = 5). respectively. In contrast, one-half of the mucinous (tumor 20). and 1 was a follicular cyst (tumor 21). The malignant tumors expressed significant amounts of amidated gastrins malignant tumors were in different stages of the disease [FIGO rating [6.7 Â±2.7 pmol/g; range, 1.4-20.0 pmol/g (n = 7)]. The concentra (21), Table 1]. In normal postmenopausal ovaria, amidated gastrins tions of glycine-extended gastrins and progastrins were low (0.25 Â± were undetectable (i.e., <0.1 pmol/g) and only low concentrations of 0.03 and 1.4 Â±0.4 pmol/g, respectively) but higher than in controls glycine extended gastrins and progastrins were measured [0.2 Â±0.05 and benign tumors (Table 1). The other half of the malignant tumors (SEM) pmol/g tissue and 0.2 Â±0.06 pmol/g, respectively (n = 4) expressed glycine-extended gastrins and progastrins [0.2 Â±0.03 and (Table 1)]. One of the five benign ovarian tumors contained a little 0.6 Â±0.1 pmol/g (n = 9)], but the amidation of the peptides was amidated gastrin (0.2 pmol/g tissue), while the concentrations of gly- impaired (0.1 Â±0.03 pmol/g). One tumor [tumor 1. (Table 1)] con- 1824

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1993 American Association for Cancer Research. GASTRIN IN OVARIAN TIMORS tained high amounts of progastrin fragment 38-54 (42.3 pmol/g be ment of the fractions with antiserum 88235. revealed two components. The minor peak eluting at A",/0.69 corresponded to processing frag fore as well as after enzymatic treatment). In all other tumors the concentration of this progastrin fragment correlated to the concentra ment 19-35 of progastrin as confirmed by calibration with synthetic tion of other gastrin components (0.1-3.8 pmol/liter). In order to progastrin 19-35. The major peak eluting at Kd 0.53 was 3 times investigate if other NHi-terminal progastrin fragments than the 38-54 higher after enzymatic treatment of the fractions (Fig. 4) than before fragment were retained in Tumor 1, we measured the tumor extract treatment (data not shown). Based on the facts that antiserum 88235 with antiserum 88235 before (14.7 pmol/g) and after (28.8 pmol/g) reacts with reduced affinity with fragments unprocessed at Arg-19 and tryptic Ã©levage,while in all other tumors these concentrations varied that the progastrin molecules in this tumor were fully processed at the between 0.7 and 4.6 pmol/g. dibasic cleavage site Arg-36/Arg-37 (Figs. 2 and 3), we assume that Gel chromatography of extracts of tumors 1 and 13 of Table 1, the immunoreactivity detected at Kti 0.53 corresponded to progastrin confirmed that the major part of the progastrin molecules were pro 1-35 or 6-35. cessed to amidated nonsulfated gastrin-17. eluting at A",/0.69. Low Neither carboxyamidated CCK, glycine-extended CCK, proCCK, amounts of gastrin-34 (A",/0.48) and progastrin (Ktl 0.20) were also nor substance P were present in detectable amounts in the tumor detected (Fig. 2; only results for tumor 1 shown; chromatography extracts. Somatostatin and prosomatostatin were undetectable, except profiles of tumor 13 were identical). in tumor 20 which contained 1.5 pmol/g tissue (wet weight). To confirm that the majority of the gastrin-17 peptides was unsulfated, tumor extracts were treated with trypsin prior to application on the column and fractions were measured using antisera 2604 and DISCUSSION 2605. when the fractions were assayed with antiserum 2604 (Fig. 3. This study has shown that ovarian serous tumors express the gastrin left), a minor peak eluting at K,, 0.56 (sulfated gastrin-17) and a major gene. All tumors and normal postmenopausal ovaria expressed pro peak eluting at K,, 0.69 (unsulfated gastrin-17) were detected. Mea gastrin, the concentration of the total gastrin mRNA product being surement with antiserum 2605 (Fig. 3, right), showed only the non sulfated component eluting at A",,0.69. The results shown for tumor 1 higher in neoplastic than in nonneoplastic tissue. The maturation of the peptides, however, differs between the tissues. The expression of are representative for tumors expressing amidated gastrins at concen gastrin is apparently selective, since other regulatory peptides such as trations of 1.4 pmol/g and higher (tumors \-4, 12-14). as judged by CCK, somatostatin, and substance P are undetectable. measurement using antisera 2604 and 2605. With the use of immunohistochemistry two studies (17. 18) found The pattern of gastrin processing in tumor 1 was confirmed by gel an increased incidence of argyrophilic cells containing gastrin immu chromatography using antisera 2145 and 88235 (Fig. 4). Tumor 1 noreactivity in mucinous ovarian tumors. This is in agreement with contained high concentrations of progastrin fragment 38-54 (eluting our observations in serous tumors. The immunohistochemistry studies at K,/ 0.58). The elution profile was identical before and after enzy used antisera, which bind both amidated gastrin and CCK. In view of matic treatment of the fractions, indicating a complete processing at our results, it is likely that the immunoreactivity is due to gastrin dibasic cleavage sites Arg 36/Arg-37 and Lys-53/Lys-54. Measure- rather than CCK. Our study demonstrates gastrin in epithelial serous ovarian tumors [the most common type of ovarian tumor (28)] and normal ovaria. All previously published case reports (7-16) and the G 17ns 750 two histopathological studies (17, 18) described gastrin synthesis only in epithelial mucinous ovarian tumors. One case study found gas trin-34 to be predominant (7), in contrast to our findings in the serous tumors, where gastrin-17 is the most abundant gastrin peptide. The detection of low amounts of progastrin and glycine-extended processing intermediates in the nonneoplastic ovaria indicates that the ovarian synthesis of gastrin is not ectopie. The total amounts of gastrin 500 expressed by the malignant ovarian tumors were high compared to the control tissues, suggesting that the expression of the gastrin gene might be coupled to cellular events involved in the transformation of the ovarian epithelium. Transcriptional activation of the gastrin gene by epidermal growth factor and transforming growth factor a, which play a role in the promotion of ovarian carcinomas (29-32) and 250 stimulate transcription of the gastrin gene (33, 34), might be respon sible for the increased synthesis. Malignant tumors containing 2 pmol/g or more gastrin were effi cient in progastrin processing. In contrast, when the amount of total gastrin was below 1.7 pmol/g tissue, maturation of progastrin was poor in both malignant and benign tumors (Fig. 5). This observation cannot be accounted for by lack of sensitivity of the assays measuring O 0.2 0.4 0.6 0.8 1 amidated gastrins, since the detection limit of these assays is 0.1 pmol/g. The processing of progastrin in the malignant ovarian tumors Elution Constant is remarkable when compared to other major epithelial cancers ex pressing the gastrin gene (Fig. 6). The processing in the antral G-cells, Fig. 2. Gel chromatography of amidated gastrins and progastrins in extract of ovarian the main site of gastrin synthesis, is efficient. The concentrations of tumor I. Crude extract of tumor I (Table I) was applied to a calibrated Sephadex G-50 superfine column ( 10 x 1000 mm) and eluted with 0.125 MNH4HCO, (pH 8.2) at 4Â°Cwith progastrin and glycine extended gastrins are, consequently, low. The a flow rate of 4 ml/h. Fractions of 1,3 ml were collected and assayed with antiserum 2604 processing in the malignant ovarian tumors resembles that of the (â€¢)and antiserum 3208 after enzymatic treatment of the fractions (â€¢). G-cells. In contrast, in bronchogenic carcinomas the processing is 1825

400 Q-17ni

300

Fig. 3. Sulfation of amidated gastrins. Trypsin treated extract of tumor I was applied to the gel filtration column (for details see legend to Fig. 2) and measured with antiserum 26Ã•W(lefÃ¬)and antiserum 2605 (righi}, s. sutfated: ns. nonsulfated.

100

Elution Constant (Kd)

100

3000 Progiitrin fragment 38-54

2500 60

2000

1500

20 1000 Total Gastrin (pmol/g)

Fig. 5. Amidation ratio versus total gastrin content in malignant and benign ovarian 500 tumors. Total gastrin content (pmol/g tissue, wet weight) of the malignant tumors (â€¢)and benign tumors (A) of Table I, plotted against the amidation ratio [amidated gastrin/total gastrin (%)].

0 0.2 0.4 0.6 0.8 1 detected in the tumor and compared to the other tumors (Figs. 2 and 4). The significance of this phenomenon is unclear. In tumor 1, a Elution Constant minor part of the progastrin was processed at the monobasic cleavage (KJ site at position Arg-19/Asp-20 of progastrin, as confirmed by gel Fig. 4. Gel chromatography of NH2-terminal gastrin processing fragments in extract of chromatography. Recently, Huebner et al. (39) characterized the pro ovarian tumor I. Conditions were identical to those described in the legend to Fig. 2. Fractions were measured with antiserum 2145 before and after trypsin (â€¢)(results were gastrin products in a pancreatic gastrinoma. Their tumor cleaved diba identical) and antiserum 88235 after tryptic cleavage of the fractions (â€¢). sic sites, but in addition it cleaved the monobasic Arg-5/Ser-6 site. The authors could not detect processing at Arg-19/Asp-20. In contrast, our relatively poor and hypergastrinemia in lung cancer has not been data support the contention that Arg-19/Asp-20 is a processing site. reported (35). Colonie carcinomas do not process progastrin (36). The nature of the gastrin synthesizing cells in the serous ovarian Gastrin is also synthesized in acoustic neuromas (37). These tumors tumors is unknown, because the gastrin concentrations were too low are rare and process progastrin poorly. Gastrinomas, also a rare type to identify the cells by immunohistochemistry. In mucinous tumors, of tumor, express high concentrations of gastrin, but the processing of however, neuroendocrine cells have been described containing, progastrin varies widely between the individual tumors (for review see among others, gastrin-like peptides and/or somatostatin (17, 18). In Ref. 37). addition, in the mucinous ovarian tumors causing the Zollinger-Elli- Tumor 1 of Table 1 displayed an unique distribution pattern of son syndrome, gastrin producing cells have been found scattered in gastrin processing fragments. The concentrations of NH2-terminal the tumor. The patchy distribution of these cells may lead to variation fragments 1-35 (or 6-35) and 38-54 of progastrin were unusually in the measurements, especially since the majority of the tumors was high in tumor 1 compared to the level of the other progastrin products large. 1826

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Involvement of transforming growth factor a/epidermal or paracrine mechanisms. Studies addressing such growth effects in growth factor receptor autocrine growth mechanism in an ovarian cancer cell line in ovarian cancer cells have now been initiated in our laboratory. vitro. Cancer Res., 51: 5951-5955. 1991. 30. Kurachi. H.. Morishige. K., Amemiya. K.. Adachi. H.. Hirota, K., Miyake. A., and Tanizawa. O. Importance of transforming growth factor a/epidermal growth factor ACKNOWLEDGMENTS receptor autocrine growth mechanism in an ovarian cancer cell line in \-i\-ti. Cancer Res., 51: 5956-5959. 1991. 31. Stromberg, K.. Collins. T. J., Gordon. A. W.. Jackson. C. L., and Johnson, G. R. The skillful technical assistance of Lis Sorensen and Kirsten Culmsee is Transforming growth factor-a acts as an autocrine growth factor in ovarian carcinoma gratefully acknowledged. We also thank Dr. Jens Knudsen for supplying tumor cell lines. 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Wouter W. van Solinge, Lars Ødum and Jens F. Rehfeld

Cancer Res 1993;53:1823-1828.

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