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Division of Cell Biology and Experimental Cancer Research, Institute of Pathology (A.F., B.W., J.C.R.), University of Bern, CH-3010 Bern, Switzerland; and Institute of Pathology (J.-O.G.), Kantonsspital Luzern, CH-6000 Luzern 16, Switzerland
Address all correspondence and requests for reprints to: Jean Claude Reubi, M.D., Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, University of Berne, Murtenstrasse 31, P.O. Box 62, CH-3010 Berne, Switzerland. E-mail: reubi{at}pathology.unibe.ch.
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Abstract |
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Objective: The objective of the study was to investigate normal and neoplastic human uterine tissues for their bombesin receptor status.
Design: In vitro subtype-specific bombesin receptor autoradiography was used in this study.
Patients: The following tissue samples were taken immediately after surgery: myometrium (n = 41), endometrium (n = 29), leiomyomas (n = 26), leiomyosarcomas (n = 6), endometrial adenocarcinomas (n = 28), and carcinosarcoma (n = 1).
Results: Normal uterine tissues expressed GRP receptors (GRP-Rs) in the myometrium, in subsets of secretory endometrial glands, and in subsets of endometrial blood vessels of the late proliferative and the secretory phase. Most leiomyomas (20 of 26) expressed GRP-R but not the leiomyosarcomas. GRP-Rs were also detected in 10 of 28 adenocarcinomas, one of one carcinosarcoma, and in blood vessels surrounding the adenocarcinomas. No other bombesin receptor subtypes (neuromedin B receptors and bb3) were detected.
Conclusions: These findings may be of physiological and pathophysiological significance. The expression of GRP-R in glands and vessels during specific phases of the cycle suggests a timely precise physiological action of GRP in these targets; in certain uterine neoplasms, the GRP-R overexpression may contribute to tumor development because GRP is a potent growth factor. Furthermore, these findings may be diagnostically and therapeutically relevant. The expression of GRP-R in leiomyomas may allow distinguishing them from receptor-negative leiomyosarcomas; GRP-R in leiomyomas, in a subset of endometrial adenocarcinomas, carcinosarcomas, and in peritumoral vessels may be candidates for receptor targeting in vivo.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Bombesin, a tetradecapeptide first discovered in the skin of the European frog Bombina bombina, together with the subsequently isolated mammalian counterparts gastrin-releasing peptide (GRP) and neuromedin B (NMB), belong to the family of bombesin-like neuropeptides (2). These peptides have been previously detected in different organs of humans and mammals, with preferential expression in nerve fibers (2). Bombesin-like neuropeptides stimulate a large number of physiological processes such as exocrine and endocrine secretion, smooth muscle contraction, and have trophic effects. These actions are mediated through specific binding of the peptides to bombesin receptors. To date, four bombesin receptor subtypes have been described: the best known and characterized, the GRP receptor (GRP-R) (BB2), the NMB receptor (BB1), and the less well-characterized bombesin receptor subtypes bb3 and bb4 (3, 4).
In the female genital tract, bombesin and GRP affect physiological functions such as contraction of the rat uterine smooth muscles (5) and growth of human endometrial stroma cells (6). Furthermore, a role of the bombesin-like peptides has been suggested in maternal, placental, and fetal development because the peptide levels are increased in fetal and maternal blood plasma and in pregnancy fluids (7, 8). Consistent with this, the GRPs and their mRNA are differentially expressed in the pregnant and nonpregnant uterus (9, 10, 11), and the peptides have been detected in various tissue compartments of the placenta (10). Immunohistochemically, the peptides have been localized in two different tissue compartments of the mammalian uterus: first, nerve fibers distributed in the myometrium and around blood vessels and, second, endometrial epithelium (11, 12). First evidence of bombesin receptors in the uterus was given by the isolation of their mRNA from the pregnant guinea pig (13) and human uterus (10, 14). The presence of the receptor protein was reported in the rat myometrium in two binding studies using 125I-Tyr4-bombesin on frozen sections (15) and on membrane homogenates (16).
Bombesin-like neuropeptides and their receptors also play a role in neoplasms. Stimulatory effects of the peptides on mitogenesis have been implicated in tumor growth of several human cancer cell lines such as lung, breast, and prostatic cancer (17). Furthermore, the corresponding receptors were detected in different human neoplasms such as breast cancer, prostate cancer, ovarian cancer, renal cell cancer, small cell lung cancer, gastrointestinal stroma tumors, bronchial carcinoids, and gastrinomas (1). Yet, in uterine neoplasms, virtually no information on bombesin receptors is available. This prompted us to investigate the expression of these receptors in normal and neoplastic human uterine tissues with in vitro autoradiography on tissue sections using 125I-Tyr4-bombesin and 125I-D-Tyr6, ß-Ala11, Phe13,Nle14-bombesin(6–14) as radioligands. Information about the incidence, distribution, density, and subtype of bombesin receptors in normal and neoplastic human uterine tissues may give insights into their physiology and pathophysiology and may allow the evaluation of the clinical value of the radiolabeled or unlabeled bombesin analogs (1, 18, 19) in this organ.
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Patients and Methods |
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Normal and neoplastic uterine tissues were taken and frozen immediately after surgical resection and stored at –70 C. The following tissues were investigated: 41 samples of normal myometrium; 29 samples of normal endometrium in various stages of the cycle and in postmenopause (10 samples of the proliferative phase, 11 samples of the secretory phase, and eight samples with endometrial atrophy); 26 leiomyomas and six leiomyosarcomas; 28 endometrial carcinomas (27 endometrioid adenocarcinomas, one clear cell adenocarcinoma); and one carcinosarcoma of the endometrium. Informed consent was obtained from the patients. The study conformed to the ethical guidelines of the Institute of Pathology in Bern and Luzern and was approved by its committees.
Receptor autoradiography
Cryostat sections (20 µm thick) of the tissue samples were processed for receptor autoradiography as described in detail previously for other peptide receptors (20). The radioligands used were 125I-Tyr4-bombesin, known to preferentially label GRP-Rs (21), and the newly developed radioligand 125I-D-Tyr6, ß-Ala11, Phe13, Nle14-bombesin(6–14), which has been reported to be an outstanding universal ligand identifying all four of the bombesin receptor subtypes (22, 23).
For autoradiography, tissue sections were mounted on precleaned microscope slides and stored at –20 C for at least 3 d to improve adhesion of the tissue to the slide. The sections were then processed as described previously (23, 24). They were first preincubated in 10 mM HEPES (pH 7.4) for 5 min at room temperature. They were then incubated in 10 mM HEPES, 130 mM NaCl, 4.7 mM KCI, 5 mM MgCl2, 1 mM ethylene glycol-bis (ß-aminoethylether)-N-N'-tetraacetic acid, 0.1% BSA, 100 µg/ml bacitracin (pH 7.4), and one of the two radioligands for 1 h at room temperature. 125I-Tyr4-bombesin (2000 Ci/mmol; Anawa, Wangen, Switzerland) was added in a concentration of 100 pM in the presence or absence of 0.1 µM bombesin. Additional sections of selected tissues were incubated in the presence of increasing amounts of nonradioactive GRP, NMB, or somatostatin (Bachem, Bubendorf, Switzerland) to generate competitive inhibition curves. The expressed bombesin receptor subtype was evaluated in the investigated samples by addition of 20 pM 125I-D-Tyr6, ß-Ala11, Phe13, Nle14-bombesin(6–14) (2000 Ci/mmol; Anawa) to the incubation solution in the absence of any competitor peptide and in the presence of 50 nM of one of the three unlabeled competitors, D-Tyr6, ß-Ala11, Phe13, Nle14-bombesin(6–14), GRP, and NMB. This protocol has been shown to discriminate adequately between the three receptor subtypes, GRP-R, NMB-R, and bb3 (23, 24). After incubation, the sections were washed four times for 2 min each in 10 mM HEPES with 0.1% BSA (pH 7.4) at 4 C. Finally, the slides were rinsed twice for 5 sec each at 4 C in HEPES without BSA. The slides were then dried under a stream of cold air. They were placed in apposition to Biomax MR films (Eastman Kodak, Rochester, NY) and exposed for 7 d in x-ray cassettes. The autoradiograms were quantified using a computer-assisted image processing system, as described previously (23, 24).
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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shows the results of GRP-R expression in normal tissues and tumors of the uterus. Normal myometrium expressed GRP-Rs in all phases of the cycle and in postmenopause, usually with moderate densities and a homogeneous distribution (Fig. 1I, A–C). In addition, a representative amount of endometrial tissue could be analyzed in 29 samples taken in various phases of the cycle, namely 10 in the proliferative phase and 11 in the secretory phase, as well as from atrophic endometrium (n = 8). Atrophic endometrium did not express detectable amounts of GRP-Rs in any component (glands, stroma, and blood vessels) (Fig. 1I, D–F). However, the functional endometrium showed a cycle-dependent expression of GRP-Rs in various tissue compartments; GRP-R expression was observed in subsets of endometrial glands in six of 11 patients, exclusively in the secretory phase (Fig. 1II). Furthermore, subsets of endometrial blood vessels expressed GRP-Rs in the muscular wall in 10 of 11 patients in the secretory phase as well as in three patients in the late proliferative phase. Figure 1III demonstrates such vessels in a uterus in the secretory phase. Single samples of functional endometrium showed tiny foci of GRP-R-positive stroma.
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). These receptors were expressed in the neoplastic tissue of 20 of 26 leiomyomas (77%). In general, the receptors were homogenously distributed, and their density was moderate to high; several samples, however, had very high density values (Fig. 2, A–E).
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Finally, we investigated 28 cases of adenocarcinomas (27 endometrioid adenocarcinomas and one clear cell adenocarcinoma) and one carcinosarcoma of the endometrium. Ten of the adenocarcinomas (all endometrioid type) expressed GRP-Rs in the neoplastic epithelial tissue compartments in a moderate density and with a heterogeneous distribution often including autoradiographically receptor-negative areas. A receptor-positive endometrioid adenocarcinoma is shown in Fig. 2, F–K. Interestingly, some adenocarcinomas expressed more GRP-Rs at the border of the tumor, namely in the tumor cells adjacent to the stroma (Fig. 3, A–C). Table 1 further shows that the tumor with the highest receptor density was a carcinosarcoma. This case is depicted in Fig. 3, D–F. A further observation made in 16 of 20 endometrial adenocarcinoma samples containing peritumoral normal uterine tissues was that small peritumoral blood vessels expressed GRP-Rs in the muscular walls (Fig. 4, A–I). These receptor-positive vessels were variable in numbers and located within a narrow rim of a few millimeters in the surrounding host tissue infiltrated by the carcinoma. Because all but one sample of leiomyomas and leiomyosarcomas contained solely tumor tissue without surrounding host tissue, they could not be assessed for the receptor status in peritumoral vessels. However, in 10 of the 26 leiomyomas and in two of the six leiomyosarcomas, scattered GRP-R-positive vessels were detected intratumorally.
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, 3, and 4); by competition experiments using 125I-Tyr4-bombesin demonstrating a strong affinity of the receptors for GRP but a weaker affinity for NMB, as shown by the complete displacement curves in Fig. 5 in three different tissues, namely myometrium, leiomyoma, and vessels; and by competition experiments with the universal radioligand 125I-D-Tyr6, ß-Ala11, Phe13, Nle14-bombesin(6–14), showing that it was well displaced by nanomolar concentrations of D-Tyr6, ß-Ala11, Phe13, Nle14-bombesin(6–14) and of GRP but only poorly with NMB (Fig. 2, A–K).
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. Normal myometrium always had a diffuse expression of a moderate density of GRP-Rs [1279 ± 164 disintegrations per minute (dpm)/mg tissue; mean ± SEM]. A subset of endometrial glands in the secretory phase expressed GRP-Rs with a density of 1411 ± 338 dmp/mg tissue (mean ± SEM), comparable with that of endometrial blood vessels of the late proliferative and secretory phases (1352 ± 128 dmp/mg tissue; mean ± SEM). The group of leiomyomas had the highest densities of GRP-Rs, followed by endometrial adenocarcinomas (2207 ± 623 and 1861 ± 472 dmp/mg tissue, respectively; mean ± SEM). The GRP-R density of peritumoral blood vessels in endometrial adenocarcinomas was similar to those of endometrial blood vessels of the late proliferative phase and the secretory phase. Finally, the case with the highest GRP-R density was a carcinosarcoma (23,502 dmp/mg tissue). |
Discussion |
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A salient finding in this study is the overexpression of GRP-Rs in most benign smooth muscle neoplasms, the leiomyomas, in contrast to their malignant counterparts, the leiomyosarcomas, which have virtually no detectable expression of these receptors. This indicates that these two tumor types have distinct biological properties with respect to GRP. Such a different biology is also in keeping with reports indicating that leiomyomas and leiomyosarcomas have distinct cytogenetic aberrations (25). Furthermore, differences between these two tumor types in the expression of numerous genes, including genes involved in metabolism and structure of cells, DNA repair, cell proliferation, and cell cycle, have been reported, suggesting distinct molecular pathways in tumorigenesis of these two types of neoplasms (26). The differential expression of GRP-Rs in benign and malignant uterine smooth muscle tumors, which is in line with these observations, could be meaningful in two respects. The overexpression of GRP-Rs in leiomyomas might play a direct pathophysiological role in growth regulation because bombesin-like peptides are known to be potent mitogens in various normal tissues and neoplasms (17); conversely, these peptides would lack any such role in leiomyosarcomas. The differential expression of GRP-Rs might be used as a diagnostic feature; radiolabeled bombesin ligands would be able to differentiate between benign and malignant uterine smooth muscle tumors.
Another important result is the detection of GRP-Rs in adenocarcinomas and carcinosarcoma of the endometrium. Together with the leiomyomas, these cancers expand the list of GRP-R-expressing human tumors (1, 23). Until now, only one study investigated functional GRP-Rs in endometrial carcinomas (27), however, not in primary cancers but only in cell lines. In the present study, we can visualize a predominantly focal expression of GRP-Rs in the epithelial compartment in nearly 40% of the endometrial adenocarcinomas. In some adenocarcinomas, the focal distribution of GRP-Rs seems to be random within the tumor tissue. In other adenocarcinomas, however, GRP-Rs are overexpressed focally in the neoplastic epithelium at the border to the surrounding tumor stroma. This may suggest that this stroma may play a role in the GRP-R regulation. Accentuated expression at the invasion front has previously been reported for different gene products including the c-met receptors in breast cancer and oral squamous carcinoma (28, 29); the c-met receptors mediate proliferative, invasive, and migratory effects of the hepatocyte growth factor. Therefore, receptor accentuation at the invasion front has been suggested to promote tumor progression (28). The same rationale might be assumed for GRP-Rs because bombesin is not only a growth factor but has also been implicated in invasion and migration of neoplastic cells (30). In addition, this specific distribution of GRP-Rs might be meaningful because it is close to the site of endogenous GRP production and secretion. Indeed, the preferential location of endogenous GRP in the uterus lies in nerve fibers (12), which are present in the surrounding preexisting tissue at the tumor border.
Another main result is the detection of GRP-Rs in the muscular layers of peritumoral blood vessels, consistent with small veins. Blood vessels overexpressing neuropeptide receptors, either veins or arteries, or both, have been previously detected by autoradiography in the immediate surroundings of different human cancers such as pancreatic ductal carcinomas (GRP-Rs) (31); gastric, breast, and prostate carcinomas (somatostatin receptors); as well as colon carcinomas (somatostatin and substance P receptors) (1). Neuropeptide receptor overexpression in peritumoral blood vessels in general and GRP-R expression in particular may be functionally meaningful because several of these neuropeptides, including substance P, somatostatin, and bombesin, are known to be vasoactive (32). They can influence regional and local blood flow via vascular tone. Endogenous neuropeptides might be present in the surroundings of peritumoral blood vessels, interact with the corresponding vascular receptor, and regulate local, peritumoral, and tumoral hemodynamics. This may apply to the GRP system as well. Indeed, not only have GRP immunoreactive nerve fibers been found around uterine blood vessels of different mammalian species (12), but bombesin has been shown to constrict vessels of humans and animals (33, 34). Therefore, the interaction of the endogenous GRP with the corresponding receptor in the muscular wall of peritumoral vessels may lead to an alterated blood flow in the tumor bed and subsequently influence tumor progression. Interestingly, GRP was also reported to cause an increase of vascular endothelial growth factor mRNA in endometrial cancer cell lines (27). Therefore, GRP may also influence angiogenesis indirectly via other growth factors and their receptors present in peritumoral vessels. Direct experimental evidence for an impact of GRP on angiogenesis has been given recently by data showing a significant decrease in the vessel density of a human experimental breast cancer in nude mice treated with bombesin/GRP antagonists (35).
This is the first study evaluating the presence of GRP-Rs at protein level in normal human uterine tissues. Various uterine tissues, namely myometrium, endometrial glands, and vessels, are able to express GRP-Rs under physiological conditions; they are the tissues of origin of the above mentioned GRP-R-expressing tumors and peritumoral blood vessels. The myometrium has GRP-Rs, an observation that is in line with pharmacological experiments in rats showing clonal contractions of isolated uterine smooth muscle tissue evoked by GRP (5). Of particular interest is, however, the timely and spatially limited GRP-R expression in different tissue compartments of the endometrium; the endometrial epithelium expresses GRP-Rs exclusively during the secretory phase and only in a subset of glands. Interestingly, previous studies with ovine endometrium taken in pregnancy (7) and during estrous cycle identified GRP and/or related peptides present in the endometrial glands; during the cycle, the GRP synthesis was intensely up-regulated during a short time of the secretory phase, the luteal regression phase (11). Together, these findings suggest a concomitant expression of GRP and its receptor in this tissue compartment, where GRP could act at least partially in an autocrine or paracrine manner. Finally, subsets of endometrial blood vessels showed a GRP-R expression restricted to the late proliferative and the secretory phase, indicating a very precise and time-dependent function of GRP on endometrial vessels as well. The exact physiological role for GRP in the endometrium, however, is still unknown (7). Particular conditions, for instance hormonal environment, might be required to stimulate GRP-R expression, conditions that are physiologically fulfilled for the glands during the secretory phase of the cycle. It remains to be investigated whether the trigger to produce GRP-Rs in endometrial tumors is the same as the one needed under physiological conditions. The spatially limited expression of GRP-Rs in the endometrial glands may perhaps be brought in relation to the focal distribution of these receptors in endometrial adenocarcinomas.
We have used two different radioligands in the present study to identify the expressed subtype of bombesin receptor. First, we used the radioligand 125I-Tyr4-bombesin that is known to bind preferentially the GRP-R subtype. Second, we used in parallel experiments the universal radioligand 125I-D-Tyr6, ß-Ala11, Phe13, Nle14-bombesin(6–14), known to label all bombesin receptor subtypes (22, 23). Displacement experiments using this radioligand with receptor subtype-selective competitors permitted the identification of a preferential expression of the GRP-R subtype in all tested tissues. However, minor amounts of other bombesin receptor subtypes cannot be completely excluded by this approach because low densities might not be detected in the presence of high densities of the predominantly expressed GRP-R (23, 24).
The present findings might be clinically relevant in several instances. Symptomatic uterine leiomyomas currently require surgical procedures. In some women, a vaginal rather than an abdominal hysterectomy is preferred but is feasible only for smaller tumors; this requires a presurgical medical treatment, for instance with gonadotropin-releasing hormone analogs (36), which induce a hypoestrogenic milieu, thus leading to a reduction of the size of the hormonally dependent leiomyomas. This strategy is, however, linked to adverse side effects such as menopausal symptoms and bone demineralization. Hence, other therapeutic strategies are being tried, such as selective estrogen receptor modulators (36) or a retinoid X receptor ligand (37), to induce shrinkage of the leiomyomas. Conceivably, GRP-Rs in uterine leiomyomas could also be targets for an adjuvant antiproliferative therapy because potent and selective GRP antagonists have been developed over the last years (19). Recently developed radiolabeled bombesin analogs (18, 38) might be used diagnostically in the discrimination of leiomyomas and leiomyosarcomas that differentially express GRP-Rs. Radiolabeled bombesin analogs (18, 38) or the cytotoxic bombesin analog AN-215 (39, 40) may also be used therapeutically in tumors with sufficient GRP-R expression; in addition to the subgroup of endometrial adenocarcinomas with a high density and diffuse distribution of GRP-Rs, the carcinosarcoma might be a potential promising candidate for targeting therapies in uterine neoplasms. This highly aggressive tumor with a poor prognosis displayed the highest GRP-R density of all tested tissues. Peritumoral blood vessels overexpressing GRP-Rs in endometrial cancers may represent attractive alternative targets for therapies aimed at influencing tumor growth through modulation of hemodynamics and angiogenesis.
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Footnotes |
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Abbreviations: GRP, Gastrin-releasing peptide; GRP-R, GRP receptor; NMB, neuromedin B.
Received May 2, 2005.
Accepted May 26, 2005.
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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), and 1000 nmol/liter somatostatin (SS-14) (
). High-affinity displacement of the tracer is found with GRP, whereas NMB induces only displacement at lower affinity. Somatostatin has no effect. Nonspecific binding was subtracted from all values. The observed rank order of potencies of these analogs is compatible with the GRP-R subtype.