Laboratoire de Communication cellulaire en Biologie de la reproduction, INSERM 407, Faculté de Médecine Lyon Sud, BP 12, F-69921 Oullins Cedex, France
Abstract
Somatostatin (SRIF) is a potent antiproliferative signal for both normal and tumoral mammalian cells and an alteration in the SRIF receptor expression pattern has been associated with carcinogenesis. In the present study, the relevance of SRIF signaling to human male germ cell tumors was assessed at the receptor level. The expression of five SRIF receptor (sst1sst5) mRNAs was estimated by RTPCR and compared between normal and tumoral testes. All 12 normal testicular tissues studied contained sst3 and sst5 receptor transcripts whereas sst4 was present in almost all (11 of 12). sst1 transcripts were consistently absent while the majority (11/12) of normal samples studied did not contain sst2 mRNA. Parallel assessment of SRIF receptor mRNAs in 10 seminoma testicular germ cell tumors showed expression of a single receptor type, sst5, in all samples analyzed. All seminoma samples were depleted in transcripts corresponding to sst1 and sst2 receptors while either sst3 or sst4 mRNAs were absent in almost all (9 of 10) tumoral samples studied. The comparison of SRIF receptor expression between normal tissue and seminoma tumors thus points to a selective loss of sst3 and sst4 mRNA expression in seminomas. Altogether these data indicate that: (i) normal human testes are putative SRIF targets; (ii) loss of sst3 and sst4 SRIF receptor expression might be associated with seminoma carcinogenesis.
Abbreviations: CNS, central nervous system; MMLV, Moloney monkey leukemia virus; SRIF, somatostatin.
Introduction
Carcinogenesis is commonly related to the abrogation of cell division control by extracellular signals such as mitogens and antiproliferative molecules (1). Besides growth factors and cytokines acting as either positive or negative regulators of cell division, another family of signaling molecules, peptide hormones, are involved in cell proliferation control. Signal transduction of peptide hormone action is generally initiated by activation of specific heptahelical transmembrane receptors. As a corollary, these receptors are possible targets for carcinogenesis and are currently considered as potential tumor markers (2).
Somatostatin (SRIF) is a peptide hormone with potent antiproliferative actions on different cell types under both physiological and pathological conditions. Additionally, SRIF is widely distributed throughout the central nervous system (CNS) and the periphery. In the CNS it fulfils criteria required for neurohormone/neurotransmitter/neuromodulator actions. In the periphery SRIF exercises antisecretory effects in many exocrine and endocrine glands. All these biological actions are mediated by five receptors (sst1sst5). They have been cloned and are encoded by different genes located on five distinct chromosomes. Almost all mammalian cells and tissues studied express multiple SRIF receptors but the combination of receptor expression is cell and tissue type specific (3).
SRIF receptors have been identified in a variety of human tumors. The most studied among them are those of neuroendocrine (e.g. GH- and TSH-producing pituitary adenomas), endocrine (e.g. enteropancreatic tumors, adrenal pheochromocytomas and medullary thyroid carcinomas) and nervous (astrocytomas, neuroblastomas and meningiomas) origin. All these tumors express more than one of the five cloned SRIF receptors (47). Their normal tissue counterparts also display multiple SRIF receptors (2). The cancer etiology is in many instances associated with an alteration in SRIF receptor expression pattern (2,8,9). Moreover, the loss of a particular SRIF receptor (i.e. sst2) has been associated with mechanisms underlying carcinogenesis in the pancreas (8,10). Otherwise, knowledge about SRIF receptor status of a given tumor is currently considered a valuable diagnostic and therapeutic criterion (11). For example, the demonstration of sst2/sst5 receptor expression in different carcinoid tumors provided the rationale for the use of synthetic SRIF agonists such as SMS201995 and BIM23014 in their clinical management (1214).
Testicular cancers are rare malignancies (37/100000) curable in up to 80% of cases in developed countries. The remaining 20% are resistant to radio- and chemotherapy for reasons that remain unknown. The incidence of testicular cancers has more than doubled over the world in the last 40 years, with a particularly strong tendency in northern and western Europe (15,16). In addition, testicular tumors of germ cell origin, representing 95% of all testicular cancers, are the commonest solid tumor in 15- to 34-year-old men and are the primary cause of death in this age group (16). Germ cell tumors are classified as non-seminomas or seminomas, each accounting for ~50% of germ cell tumors. Pathogenetic mechanisms leading to the appearance of either of them are unknown (15). In particular, the absence of germ cell lines render such mechanisms difficult to study.
SRIF has been found in both normal human testes (17) and testicular cancers of germ cell origin (18). However, previous studies addressed only sst5 SRIF receptor expression in normal human testes (19), whereas a possible alteration in receptor expression has not been assessed in these pathologies before.
In the present work we therefore compared testicular SRIF receptor mRNA expression between samples obtained from normal testes and seminomas using the RTPCR approach. Our data point to differential expression of sst1sst5 receptors between the two experimental groups.
Materials and methods
Subjects and tissue samples
Ten patients with testicular cancer of pure seminoma type, not metastatic to supradiaphragmatic nodal or visceral sites, were included in this study. The seminoma samples used were of stage I and II (disease limited to either testis, epididymis, spermatic cord or retroperitoneal lymph nodes, respectively). Biopsies of surrounding testicular tissues with histologically normal appearance were considered as normal controls. In addition, two subjects with histologically normal testes that had been orchiectomized after diagnosis of prostate cancer were also added to the experimental cohort. The patient age ranged from 26 to 56 years (mean 34.5). All testicular samples were collected by the Department of Pathology, Antiquaille Hospital, Lyon, France. Informed consent of the patients was obtained and approved by the Board of the Hospital.
At surgery, tissue samples were divided into a few fragments: they were randomly either frozen and stored in liquid nitrogen for RTPCR or fixed in 4% paraformaldehyde for histopathological analysis. Clinical diagnoses of seminomas (including serum negativity for -fetoprotein) were confirmed cytologically based on the presence of immunoreactivity for placental alkaline phosphatase. Only specimens containing 100% normal or seminoma cancer cells were retained for study.
RNA extraction and RTPCR conditions
TRIzol reagent (Life Technologies, Eragny, France) was used to extract total cellular RNA and DNA from tissue samples. In order to exclude any genomic DNA contamination, 10 µg of each RNA sample were treated for 10 min at 37°C with 0.75 U DNase I (Pharmacia Biotech, Uppsala, Sweden) in the presence of 40 U RNase inhibitor, RNasin (Promega, Charbonnières, France), 5 mM DTT (Gibco BRL, Cergy Pontoise, France) and buffer 1X (Gibco BRL) in a final volume of 10 µl. The reaction was terminated by heating at 75°C for 10 min. Treated RNA was reverse transcribed with Moloney monkey leukemia virus (MMLV) reverse transcriptase (200 U) in a reaction tube containing 0.5 mM each deoxynucleotide triphosphate (Pharmacia Biotech), 40 U RNasin (Promega), 5 µM hexanucleotide primer (Sigma, l'Isle d'Abeau, France), 10 mM DTT (Gibco BRL) and buffer (Gibco BRL) in a final volume of 20 µl. The mixture was first incubated at 42°C for 1 h and subsequently boiled for 5 min. Then 2 µl of the RT reaction mixture from each sample were diluted into a final volume of 50 µl in 50 mM TrisHCl buffer (Promega) containing 0.2 mM dNTP (Pharmacia Biotech), 1 U Taq DNA polymerase (Promega) and 0.4 µM each primer (Table I).
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Results
Clinicopathological data on tissue samples used in the present study are given in Table II. The results shown in Figures 15
correspond to specimens N1N6 for normal tissue and S1S6 for seminomas that were obtained from cases 16 in Table II
. Results corresponding to specimens N7N12 and S7S10 (Table II
) are not presented in the figures; they are summarized in Table III
.
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SRIF receptor mRNAs were detected in all normal and tumor testicle samples studied. Normal tissues express mRNAs for more than one receptor type (Figures 35). In contrast, in seminomas, only expression of sst5 receptor mRNA was consistently observed (Figure 5
).
Concerning individual receptor transcripts, sst1 was absent in all normal samples (corresponding to specimens N1N12 obtained from cases 112 in Table II; results corresponding to the first six specimens are shown in Figure 1
) whereas sst2 mRNA was absent in 11 of 12 samples. The only normal testicle sample positive for sst2 receptor expression came from case number 6 (specimen N6, Table II
and Figure 2
, lane 24). However, the selected sst1 and sst2 primers amplified fragments of the expected size from genomic DNA used as a positive control (Figures 1 and 2
, lanes 3 and 18). Moreover, in co-amplification reactions performed with sst2 and ß-actin primers, a product corresponding to the latter was systematically found (Figure 2
). All normal testicle samples tested contained sst3 and sst5 mRNA. The sizes of fragments amplified from cDNA were identical to those obtained with genomic DNA (positive control) used as template; fragments corresponding to ß-actin were co-amplified in all cases (Figures 3 and 5
). Similarly, 11 of 12 normal testicle samples (specimens N1 and N3N12 in Table II
, of which the first six, N1N6, are shown in Figure 4
) display sst4 transcripts; the sole sst4 mRNA-negative sample (N2) corresponds to case number 2 (Table II
and Figure 4
, lane 7).
No amplification product was obtained with primers specific for sst1 and sst2 when seminoma cDNA was used as template (corresponding to specimens S1S10 in Table II; results corresponding to the first six samples are shown in Figures 1 and 2
). Nevertheless, the internal standard (a fragment corresponding to ß-actin) for co-amplification of sst2/ß-actin was visualized in all samples (Figure 2
, lanes 11, 13, 15, 26, 28 and 30). All of 10 seminoma samples tested expressed sst5 receptor mRNA (Figure 5
, lanes 11, 13, 15, 26, 28 and 30, corresponding to specimens S1S6 in Table II
). Indeed, a 154 bp long product was obtained in PCR assays using either genomic or cDNA as template.
The most striking difference between normal and tumoral samples was seen in relation to the expression of sst3 and sst4 receptor transcripts. They were lost in 90% of tumors, contrasting with their presence in >90% of normal samples (shown in Figures 3 and 4 for the first six specimens presented in Table II
). The absence of PCR products corresponding to these two receptors is not due to inappropriate experimental conditions since fragments of the expected size were amplified from a genomic DNA (positive control) template for both sst3 and sst4 (Figures 3 and 4
, lanes 3 and 18). In addition, in co-amplification reactions of sst3/ß-actin all seminoma samples displayed the PCR products corresponding to ß-actin (Figure 3
, lanes 11, 13, 15, 26, 28 and 30). The only seminoma samples expressing either sst3 (Figure 3
, lane 28) or sst4 (Figure 4
, lane 11) receptor mRNAs came from two different subjects corresponding to specimens S5 and S1 obtained from cases number 5 and number 1, respectively (Table II
).
The expression of different SRIF receptors is summarized in Table III.
Discussion
The results presented in this paper point to a selective loss of sst3 and sst4 receptor expression in testicular germ cell cancer of seminoma type. However, sst3 and sst4 transcripts are each expressed in one of 10 seminoma samples tested. Since there was no amplification in RTPCR reactions in which either template or MMLV reverse transcriptase was ommitted (negative controls), the observed fragments corresponding to sst3 and sst4 receptors obtained with these two seminomas could not be the result of amplification of contaminating genomic DNA sequences. They are rather due to the well-documented inter-individual variability in SRIF receptor expression by tumors (2). Alternatively, these two samples might have come from two subjects in whom the cancerization process was less advanced than in the remaining nine subjects considered in our study. Indeed, the two cases (case number 5 for sst3 and case number 1 for sst4) are the only two patients in our experimental cohort who displayed a tumor stage I seminoma with a maximal tumor diameter 2 cm.
In addition, our data show sst3, sst4 and sst5 SRIF receptor mRNA expression in normal human testes. Besides, expression of SRIF receptor mRNA is variable to some extent in normal testes as well. For example, the sst2 transcript was present in one normal sample whereas it was lacking in 11 other samples tested. The opposite was observed for sst4 receptor mRNA. In contrast, expression of sst5 and absence of sst1 receptor mRNA expression appear much less polymorphous in both normal and seminoma samples.
Given the heterogeneous SRIF receptor expression in different tumor types (for a review see ref. 2), it is not possible to extrapolate the loss of sst3 and sst4 receptor mRNA expression observed here in testicular cancer to the general pathogenetic mechanisms of carcinogenesis. Nevertheless, at this point it is tempting to speculate on an emerging common tendency concerning these receptors. Thus, as in seminomas, in all tumors in which the expression pattern of SRIF receptors has been estimated, the sst3 receptor was rarely expressed or absent. Indeed, small cell lung carcinoma (22), pancreatic, colorectal (8) and prostate (9) cancers as well as pituitary adenoma (4) lack this receptor type. In addition, in accordance with our data, the sst4 transcript was not found in pituitary adenoma (4) and medullary thyroid carcinoma (7). However, it seems clear that, if relevant, such an absence or loss of sst3 and sst4 receptor expression cannot be considered as the sole mechanism involved in carcinogenesis. For example, in pancreatic carcinomas which lack the sst3 SRIF receptor, carcinogenesis was formally associated with loss of sst2 receptor expression. In an elegant study, Delesque and colleagues demonstrated that re-expression of the sst2 receptor reverses tumorigenecity (10). Further studies on additional tumor types are now needed in order to extend these findings.
The physiological significance of the presence of SRIF receptors in normal human testis as well as the pathological implications of their observed loss in seminoma remains to be elucidated. However, it is of interest to note the observations made in a previous work in which SRIF analog (SMS201995) injection was used as a tool to measure growth hormone secretion recovery in healthy adult males during physical exercise. This study reported an unexpected, rapid (2 h after SMS201995 injection) rise in serum testosterone level in both rest and exercise trials. Such an increase in testosterone secretion occurred without a simultaneous increase in LH secretion, prompting the authors to suggest that SRIF can modulate testosterone secretion at the testicular level (23). Our data concerning sst3 and sst5 SRIF receptor expression in normal human testes are compatible with the actions of SMS201995 in vivo. Indeed, this analog acts on human sst2, sst3 and sst5 receptors (24). Similar conclusions on SRIF-mediated regulation of testosterone secretion have also been documented in animal models (25). The presence of SRIF and its receptors in human (17; present study) and animal (26,27) testes therefore supports the existence of auto/paracrine loops controling local testosterone secretion. In the light of this and in order to advance our understanding of SRIF testicular functions, a systematic evaluation of male gonadal parameters as part of a routine survey of patients suffering from neuroendocrine tumors and undergoing long-term treatment with stable SRIF analogs might produce precious information.
Otherwise, concerning direct SRIF effects on germ cell proliferation, our data on the loss of SRIF receptor expression in seminomas (characterized by uncontrolled, high rate germ cell proliferation) are compatible with the proposed antiproliferative actions of this peptide. However, the possible negative regulation of germ cell proliferation by SRIF, such as has been documented in other cell types (3), remains to be demonstrated explicitly in human and animal testicular models. The establishment of germ cell lines as paradigms to study physio-pathological actions of SRIF in mammalian testes will probably help our understanding of underlying mechanisms.
In conclusion, our data indicate that a routine RTPCR assessement of SRIF receptor expression in testicular biopsies may turn out to have important diagnostic and therapeutic implications. However, further studies at the international level are now required in order to confirm on a larger scale the data reported here on a relatively modest tumor cohort issuing from a low (but increasing) frequency of seminoma. If confirmed by other studies, our data would suggest that a comparison of SRIF receptor expression patterns in tumoral and surrounding normal testicular tissues revealing a loss of particular receptor types might help to establish seminoma diagnosis. Knowledge of receptor expression patterns could also be used to develop clinical trials for testing synthetic SRIF analogs in germ cell tumor therapies. Our results show that seminomas express the sst5 receptor, which can be targeted with high affinity by stable SRIF agonists (e.g. SMS201995). This analog is already used in the clinical treatment of a number of tumors (for a review see ref. 2). It is now worthwhile to compare the potential therapeutic benefit of SRIF analogs with those provided by classical radio- and chemotherapies of seminomas.
Acknowledgments
We are greatly indebted to Drs R.Bouvier (Edouard Herriot Hospital, Lyon, France) and N.Dutrieux-Berger (Antiquaille Hospital, Lyon, France) for the normal and seminoma testicular sample supplies and histopathology. We should like to thank Mrs Monique Billaud (Médiathèque-Santé) for expert graphical work and Mrs Glynis Thoiron for the English revision. This work was supported by ARC grant 7244 (S.K.) and Ligue contre le Cancer: Comité de l'Ardèche (S.K.) and Comité de l'Ain (M.B.).
Notes
1 To whom correspondence should be addressed Email address: krantic{at}lyon151.inserm.fr
References