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Original Article |
Endocrine, Polypeptide, and Cancer Institute, Veterans Affairs Medical Center, and Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana 70112
Address all correspondence and requests for reprints to: Dr. Andrew V. Schally, Veterans Affairs Medical Center, 1601 Perdido Street, New Orleans, Louisiana 70112-1262.
Abstract
Antagonists of GHRH inhibit the growth of various human tumors, including prostate cancer, but the tumoral receptors mediating the antiproliferative effect of GHRH antagonists have not been clearly identified. Recently, we demonstrated that human cancer cell lines express splice variants (SVs) of receptors for GHRH, of which SV1 exhibits the greatest similarity to the pituitary GHRH receptors. In this study we investigated the expression of GHRH and SVs of GHRH receptor and the binding characteristics of the GHRH receptor isoform in 20 surgical specimens of organ-confined and locally advanced human prostatic adenocarcinomas. The mRNA expression of GHRH and SVs of GHRH receptor was investigated by RT-PCR. The affinity and density of receptors for GHRH were determined by ligand competition assays based on binding of 125I-labeled GHRH antagonist JV-1-42 to tumor membranes. Twelve of 20 tumors (60%) exhibited specific, high affinity binding for JV-1-42, with a mean dissociation constant (Kd) of 0.81 nmol/liter and a mean maximal binding capacity of 185.2 fmol/mg membrane protein. The mRNA of SV1 was detected in 13 of 20 (65%) prostate cancer specimens and was consistent with the presence of GHRH binding. RT-PCR analyses also revealed the expression of mRNA for GHRH in 13 of 15 (86%) prostatic carcinoma specimens examined. The presence of GHRH and its tumoral receptor SVs in prostate cancers suggests the possible existence of an autocrine mitogenic loop. The antitumor effects of GHRH antagonists in prostate cancer could be exerted in part by interference with this local GHRH system.
CARCINOMA OF THE PROSTATE is the most frequently diagnosed noncutaneous malignancy and the second leading cause of cancer-related deaths among men in the U.S. (1). The present modalities of treatment for advanced prostate cancer are palliative and based upon androgen deprivation (2, 3, 4). However, after a period of remission most patients eventually relapse and die of androgen-independent prostate cancer. Thus, new therapeutic approaches have to be developed for the management of relapsed androgen refractory prostate cancer (2, 3, 4).
Recently, we synthesized potent antagonistic analogs of GHRH for the treatment of various cancers (5). GHRH antagonists strongly suppressed the in vivo growth of various experimental cancers, such as prostatic (6, 7), mammary (8, 9, 10), and ovarian (11) cancers; renal cell carcinomas (12, 13); small cell lung cancers (SCLC) and non-SCLC (14, 15); pancreatic (16) and colorectal carcinomas (17); bone sarcomas (18, 19); and malignant glioblastomas (20). The antitumor effects of GHRH antagonists were initially thought to be exerted only indirectly through inhibition of the endocrine pituitary GH/hepatic IGF-I axis and the reduction in serum IGF-I levels (5). However, inhibition of the proliferation of various human cancer cell lines cultured in vitro and suppression of the production of IGF-II suggested that the antagonists of GHRH must also exert some direct effect on tumors (6, 7, 8, 11, 12, 14, 16, 17, 18, 19, 20, 21). These findings together with the reduction in the tumoral concentrations of IGF-I and IGF-II and the suppression of the gene expression of IGF-I and IGF-II, in various cancers in vivo in the absence of any significant effect on serum IGF-I led to the conclusion that GHRH antagonists exert a direct action on tumor growth (5, 22). Such an action would have to be mediated through specific GHRH receptors on tumors (5, 22, 23). Although in initial studies the pituitary form of GHRH receptors could not be detected in any of the cancer models (5, 8, 22, 23), very recent investigations demonstrated that various human experimental cancers, including LNCaP, DU-145, PC-3, ALVA-41, and MDA-PCa-2b prostatic carcinomas, express splice variants (SVs) of GHRH receptors (9, 11, 13, 24, 25, 26, 27). The isolation and sequencing of cDNA encoding the SVs of GHRH receptors on these human tumors showed that they are distinct from the pituitary GHRH receptors (13, 24). Among these truncated forms of GHRH receptor, SV1 displays the greatest similarity to the pituitary GHRH receptor and is predominantly detected in the human experimental tumor models tested. Using 125I-labeled GHRH antagonist JV-1-42 as a new radioligand, we were also able to detect specific, high affinity binding sites for GHRH and its antagonists in several cancer models investigated (9, 11, 13, 24, 27).
To confirm that the GHRH antagonist JV-1-42 binds to the GHRH receptor isoform encoded by SV1 and to ascertain whether SV1 mediates mitogenic effects on nonpituitary tissues, we expressed SV1 in 3T3 mouse fibroblasts and studied the properties of the transfected cells (28). Radioligand binding assays with 125I-labeled JV-1-42 detected high affinity binding sites on 3T3 cells transfected with pcDNA3-SV1, whereas the control cells transfected with the empty vector (pcDNA3) did not show any binding (28). Cell proliferation studies showed that cells expressing SV1 are much more sensitive to GHRH analogs than pcDNA3 controls (28). Thus, the expression of SV1 augments the mitogenic responses of 3T3 cells to GHRH-(1–29)NH2 or GHRH agonist JI-38 and the antimitogenic responses to GHRH antagonist JV-1-38 compared with those of control cells not expressing SV1 (28). In addition, we found that the stimulation of SV1-transfected 3T3 cells by GHRH or its agonist JI-38 induces the production of cAMP, in contrast to control cells, which do not show a cAMP response (28). These results strongly suggest that the gene product of SV1 is a functional receptor that relays mitogenic and other signals in response to GHRH. GHRH antagonists can counteract the effects of GHRH through a competitive binding to the SV1 isoform of GHRH receptor (28). These studies support the concept that such tumoral GHRH receptors might mediate the mitogenic effect of GHRH, which was previously shown to be produced locally by various human cancers, such as endometrial, ovarian, breast, and pancreatic, and SCLC cell lines (15, 29, 30, 31).
Although the expression of SVs of GHRH receptors has now been reported in several human cancer models, little is known about the nature and expression pattern of these tumoral receptors in primary human tumors. To address this issue, we investigated in the present study the expression of SVs of GHRH receptors and the binding characteristics of the GHRH receptor isoform in 20 specimens of organ-confined and locally advanced prostate cancers obtained after radical prostatectomy. In addition, we evaluated the expression of mRNA for GHRH as well as its incidence.
Materials and Methods
Peptides and chemicals
Human (h) GHRH-(1–44)NH2 and GHRH antagonists JV-1-42 ([PhAc-His1, D-Arg2, Phe(4-Cl)6, Arg9,Abu15, Nle27, D-Arg28,Har29]hGHRH-(1–29)NH2), JV-1-36, and JV-1-38 as well as vasoactive intestinal peptide (VIP) antagonist JV-1-53 were synthesized by solid phase methods, and purified and analyzed as described previously (32, 33). In JV-1-42, PhAc is phenylacetyl, Phe(4-Cl) is 4-chlorophenylalanine, Abu is 
-aminobutyric acid, Nle is norleucine, and Har is homoarginine. Agonistic analog [His1,Nle27]hGHRH-(1–32)NH2, VIP, and pituitary adenylate cyclase-activating polypeptide (PACAP) were obtained from California Peptide Research, Inc. (Napa, CA). Potent VIP1 receptor-specific antagonist (PG 97–269) was provided by Drs. P. Gourlet and P. Robberecht (Université Libre, Brussels, Belgium) (34). Radioisotope 125I-labeled sodium was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). All other peptides and chemicals, unless otherwise mentioned, were obtained from Sigma (St. Louis, MO), Bachem (Torrance, CA), and R\|[amp ]\|D Systems, Inc. (Minneapolis, MN).
Radioiodination
Radioiodinated derivatives of GHRH antagonist JV-1-42 were prepared by the chloramine-T method as previously described (35) with some modifications. Briefly, 10 µg JV-1-42 dissolved in 10 µl 0.01 mol/liter aqueous acetic acid were added to 80 µl 0.1 mol/liter ammonium acetate in 50% (vol/vol) aqueous acetonitrile (pH 6.5–7) in a borosilicate glass tube. Radioiodination was carried out with approximately 2 mCi Na125I for 45 sec at room temperature. The reaction was initiated with 10 µg chloramine-T in 10 µl 0.1 mol/liter ammonium acetate in 50% (vol/vol) aqueous acetonitrile (pH 6.5–7) and was terminated by adding 50 µg L-cysteine in 10 µl 0.01 N aqueous HCl. The labeled peptide was purified by reverse phase HPLC on a Vydac 214TP52 column (250 x 2 mm, with C4 packing; Vydac, Hesperia, CA), using 0.1% (vol/vol) aqueous trifluoroacetic acid as solvent A and 0.1% trifluoroacetic acid in 70% (vol/vol) aqueous acetonitrile as solvent B. Elution was carried out by a linear gradient of 45–65% solvent B in 50 min. The effluent was monitored by a UV detector at 215 nm and a flow-through radioactivity detector constructed from a ratemeter (SML-2, Technical Associates, Canoga Park, CA) in our laboratory. Four hundred-microliter fractions were collected in polypropylene tubes, each containing 30 µg bacitracin and 10 mg BSA dissolved in 1 ml 50 mmol/liter Tris buffer (pH 7.4). The fractions, corresponding to the monoiodinated compound and identified by elution position, radioactivity, and UV peak, were stored at -70 C for the in vitro receptor studies. As phosphate buffer precipitates JV-1-42, the use of this buffer was avoided during radioiodination as well as during collection and storage of 125I-labeled fractions of JV-1-42. Radioiodinated derivatives of [His1,Nle27]hGHRH-(1–32)NH2 were prepared as described previously (35).
Tissue samples from patients
Twenty human prostate cancer specimens were obtained from patients, 48–82 yr of age (mean age, 63.1 ± 8.1 yr) at the time of initial surgical treatment at the V.A. Medical Center (New Orleans, LA). The local internal review board approved the collection and use of these specimens for these studies. All analyses were first conducted to meet the primary clinical requirement for patient management, and only residual tissue was used for this study. After surgical removal, select portions of the prostate cancer tissues were flash-frozen in liquid nitrogen and taken on dry ice, together with the pathology reports, to our laboratory at the V.A. Medical Center, where all the receptor analyses and molecular biology studies were performed. Histopathological examination of each specimen was undertaken to confirm the presence of cancer with minimal admixed nonmalignant tissue (<20%) before the receptor studies. All cancer samples were primary tumors, not metastases. Clinical and pathological data are shown in Tables 1
and 2. The intact specimens and their membrane fractions were stored at -70 C until analyses of GHRH-binding sites and molecular biology studies. The expression of SVs of GHRH receptor and the binding characteristics of the GHRH receptor isoform were investigated in all 20 surgical specimens of human prostate adenocarcinomas, but due to the limited amounts of RNA, the expression of mRNA for hGHRH was examined in only 15 specimens.
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Preparation of membranes for receptor studies was performed as described previously (13, 35). Briefly, the samples were thawed and cleaned, and then the specimens were homogenized in 50 mmol/liter Tris-HCl buffer (pH 7.4), supplemented with protease inhibitors (0.25 mmol/liter phenylmethylsulfonylfluoride, 2 µg/ml pepstatin A, and 0.4% aprotinin) using an Ultra-Turrax tissue homogenizer (IKA Works, Wilmington, NC) on ice. The homogenate was centrifuged at 500 x g for 10 min at 4 C to remove the nuclear debris and lipid layer. The supernatant containing the crude membrane fraction was ultracentrifuged (L8-80M, Beckman, Palo Alto, CA) twice at 70,000 x g for 50 min at 4 C after resuspending in fresh buffer. The final pellet was resuspended in homogenization buffer and stored at -70 C until assayed. Protein concentrations were determined by the method of Bradford using a protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA).
GHRH receptor binding assays were carried out as reported in detail, using in vitro ligand competition assays based on binding of [125I]JV-1-42 as radioligand to tumor membrane fractions (13). In brief, membrane homogenates containing 50–160 µg protein were incubated in duplicate or triplicate with 60,000–80,000 cpm [125I]JV-1-42 and increasing concentrations (10-12–10-6 mol/liter) of nonradioactive peptides as competitors in a total volume of 300 µl binding buffer (50 mmol/liter Tris-HCl, 5 mmol/liter EDTA, 5 mmol/liter MgCl2, 1% BSA, and 30 µg/ml bacitracin, pH 7.4) supplemented with protease inhibitors as mentioned above. Binding reactions were performed in siliconized polypropylene tubes (Sigma), where the nonspecific binding of [125I]JV-1-42 to the assay tubes was less than 5% (13). After 1 h of incubation at room temperature, the tubes were immersed to ice water, 250 µl of the suspension were transferred to cold siliconized polypropylene microfuge tubes (Sigma) and centrifuged at 19,000 x g for 2 min (Beckman J2-21M) at 4 C, and the supernatant was removed by aspiration. The pellet was washed twice with 500 µl ice-cold binding buffer, and then the bottoms of the tubes, containing the pellet, were cut off and counted in a 
-counter (Micromedic Systems, Huntsville, AL).
RNA isolation
Total RNA from human prostate cancer specimens was extracted according to the Tri-Reagent protocol (Sigma) as previously described (36). Polyadenylated RNA was purified from total RNA using oligo(deoxythymidine)-cellulose (MicroPolyAPure mRNA Isolation Kit, Ambion, Inc., Austin, TX) (24). The concentration of mRNA was determined by spectrophotometric analysis at A260/280nm.
RT-PCR analyses of mRNA expression of hGHRH and hGHRH receptor splice variants
One microgram of polyadenylated RNA was subjected to RT reaction and then amplified using the reagents and protocol of the GeneAmp RNA PCR Core kit (Perkin-Elmer, Norwalk, CT). The RT reaction was performed in a final volume of 20 µl containing 2.5 µmol/liter oligo(deoxythymidine), 1 mmol/liter each of the deoxy-NTPs, 1x PCR buffer II, 5 mmol/liter MgCl2, 1 U/µl ribonuclease inhibitor, and 2.5 U/µl Moloney murine leukemia virus reverse transcriptase. Eight or 5 µl of the RT reaction were used for each PCR amplification with primer sets that would amplify cDNAs for hGHRH (15) or hGHRH receptor SVs (24). The PCR reaction included 1x PCR buffer II, 2 mmol/liter MgCl2, 0.75 µmol/liter for GHRH or 1.0 µmol/liter for GHRH receptor SVs of each primer, and 2.5 U AmpliTaq Gold DNA polymerase for GHRH or AmpliTaq DNA polymerase for GHRH receptor SVs in a 25-µl volume. The PCR amplification was performed in a GeneAmp PCR System 2400 (Perkin-Elmer) with the following cycle profiles: 95 C for 10 min, followed by 45 cycles of 95 C for 30 sec, 55 C for 30 sec, and 72 C for 30 sec for GHRH or 95 C for 180 sec, followed by 45 cycles of 95 C for 30 sec, 60 C for 30 sec, and 72 C for 45 sec for GHRH receptor SVs. After the last cycle, there was a final extension for 7 min at 72 C. In the case of GHRH receptor SVs, the primary PCR product was diluted 1:50 with Tricine-EDTA buffer, and a secondary PCR with 20 cycles was subsequently performed using 5 µl of the primary PCR product and 1.0 µmol/liter of each nested primer (24) in a total volume of 25 µl with the same cycle profile as described above. The final PCR products were subjected to electrophoresis on a 1.8% agarose gel, stained with 0.5 µg/ml ethidium bromide, and visualized under UV light, followed by scanning of the gel using a GDS 7500 Gel Documentation System (UVP, Upland, CA) and a GS-700 Imaging Densitometer (Bio-Rad Laboratories, Inc.). The quality of the RNA extracted was tested by PCR amplification of human ß-actin cDNA (36) from the same RT reaction used for cDNA amplifications described above. A reverse transcriptase negative reaction was also performed with a mixture of RNAs from various human prostate cancer specimens as the negative control.
Analysis of experimental data
Specific ligand binding capacities and affinities were calculated by the Ligand-PC computerized curve-fitting program developed by Munson and Rodbard (37). To determine the types of receptor binding, equilibrium dissociation constants (Kd values), and the maximal binding capacity of receptors (Bmax), GHRH binding data were also analyzed by the Scatchard method (38). Statistical analyses were performed using SigmaStat software (Jandel, San Rafael, CA). A Spearman rank order correlation test was used to evaluate the correlation coefficients, and a Fisher exact test was performed to compare the incidence of GHRH receptor expression in various groups.
Results
Radioligand binding studies
The presence of specific GHRH-binding sites and characteristics of binding of 125I-labeled GHRH antagonist JV-1-42 to membrane receptors on human prostatic adenocarcinomas were determined using ligand competition assays. Of the 20 tumor preparations examined, 12 showed GHRH receptor binding (60%; Table 1
). Analyses of the typical displacement of radiolabeled JV-1-42 by the same unlabeled peptide revealed that the one-site model provided the best fit, indicating the presence of one class of high affinity GHRH receptors in crude membranes of human prostate cancer specimens. The computerized nonlinear curve fitting and the Scatchard plot analyses of the binding data (Fig. 1) in 12 receptor-positive tumor specimens indicated that the single class of high affinity binding sites had a mean Kd of 0.81 ± 0.26 nM (range, 0.36–1.25 nM), with a mean Bmax of 185.2 ± 78.7 fmol/mg membrane protein (range, 88.7–305.2 fmol/mg protein). Biochemical parameters essential to establish the identity of specific binding sites were also determined. Thus, the binding of 125I-labeled JV-1-42 in human prostate cancer specimens examined was found to be reversible, time and temperature dependent, and linear with protein concentration (data not shown). The specificity of GHRH binding was demonstrated by competitive binding experiments using several peptides structurally related or unrelated to GHRH (Fig. 2). The binding of radiolabeled JV-1-42 was completely displaced by increasing concentrations (10-12–10-6 M) of GHRH and its agonistic or antagonistic analogs, whereas peptides of the VIP-glucagon-PACAP family sharing structural homology with hGHRH did not inhibit the binding of radioligand JV-1-42. In addition, none of the structurally and functionally unrelated peptides tested, such as LH-releasing hormone, somatostatin, epidermal growth factor, [Tyr4]bombesin, and IGF-I, competed with radiolabeled JV-1-42 at concentrations as high as 1 µM (Fig. 2).
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PCR analysis of expression of mRNA for hGHRH in prostate cancer specimens
RT of RNA, followed by PCR amplification with primers specific for human GHRH, yielded a product of the expected size (322 bp) in the positive control (H-69 human SCLC; Fig. 3). A PCR product of the same molecular size was observed in 13 of 15 prostate cancers examined (87%; Fig. 3 and Table 1).
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The expression of tumoral GHRH receptor SVs was assessed by nested PCR. Using primers corresponding to intron 3 and exon 12 of the hGHRH receptor gene in the primary PCR, and primers designed for intron 3 and exon 8 in the secondary PCR, we were able to amplify 720-, 566-, and 335-bp PCR products in various proportions in human prostate cancer specimens (Fig. 4). Sequence analyses of these three PCR fragments revealed different major open reading frames, which corresponded to SV1, SV2, and SV4 of the GHRH receptor described previously (24). Bands representing SV1 were detected in 13 of 20 cancers examined (65%), whereas the incidence of SV2 and SV4 was 60% and 15%, respectively (Table 1 and Fig. 4). No PCR products for SV3 were obtained in any of the 20 human prostate cancer specimens tested. The expression pattern of these SVs as well as receptor binding characteristics and clinical and pathological data in 20 prostate cancer specimens are shown in Table 1.
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and Fig. 4). Seven of the 20 prostate cancers did not exhibit mRNA for SV1 or show ligand binding; conversely, all receptor-positive specimens expressed a detectable amount of SV1. Two samples showed neither JV-1-42 binding nor the expression of mRNA for hGHRH or SV1 (Table 1). However, primers designed for cDNA of human ß-actin were able to amplify a 459-bp PCR product from the same RT reaction, proving the proper integrity of the RNA isolated from these tumor specimens. Correlation of SVs of GHRH receptor with clinical and pathological findings
Table 2 shows GHRH receptor status in relation to cancer grade (Gleason score) and pathological stage in 20 human prostate cancers. In a subgroup of patients at high risk for recurrent cancer (stage pT3 and/or Gleason score of 8–10), the incidence of SV1 expression and JV-1-42 binding was higher than that observed in the low risk group (Table 2).
There was no correlation between receptor binding characteristics and pathological stage or cancer grade. In addition, the presence or frequency of expression of SV1 in prostate cancers was not related to the patient’s age. Finally, no correlation was observed between the patient’s age and the concentration (Bmax) or affinity (Kd) of GHRH binding sites.
Discussion
New therapeutic modalities are needed to improve the management of relapsed androgen-independent prostate cancers. Recently, we synthesized potent antagonists of GHRH for treatment of a wide range of neoplasms potentially dependent on IGF-I, IGF-II, and GHRH (5, 22). Numerous studies showed that GHRH antagonists inhibit the growth of various human cancer cell lines xenografted into nude mice, including androgen-independent human PC-3 and DU-145 prostate cancers and Dunning R-3327 AT-1 rat prostatic adenocarcinoma (6, 7). Accumulating evidence suggests that in addition to inhibiting the pituitary GH/hepatic IGF-I axis, GHRH antagonists may exert direct antitumor effects on various cancers mediated through specific receptors (5, 7, 22, 23, 36). Nevertheless, earlier studies based on approaches such as RT-PCR and radioreceptor assays did not detect the expression of the pituitary form of GHRH receptors in the cancer models examined (8, 11, 13, 22, 23, 36). Very recently we reported that various cancer cell lines, including LNCaP, DU-145, PC-3, and MDA-PCa-2b prostatic, MiaPaCa-2 pancreatic, CAKI-1 renal, MDA-MB-435 breast, H-69 SCLC, OV-1063 ovarian, and MNNG/HOS and SK-ES-1 bone sarcomas, express SVs of GHRH receptors, encoding an alternate form of the GHRH receptor (9, 11, 13, 24, 25, 27). Subsequently, Chopin and Herington (26) also found SV-I of GHRH receptors in ALVA-41, LNCaP, DU-145, and PC-3 models of prostate cancer. However, little is known about the nature and expression pattern of these tumoral receptors in human primary tumors.
This study reports the existence of GHRH-binding sites in specimens of organ-confined and locally advanced prostate cancers obtained after radical prostatectomy. Using 125I-labeled GHRH antagonist JV-1-42 as a radioligand, we were able to demonstrate the presence of specific, high affinity binding sites for GHRH and its antagonists in membrane fractions of primary prostate tumors. Our results indicate that 60% of the human prostatic carcinomas possess specific, high affinity GHRH receptors. The density of the receptors showed slight intersubject variability, but the average concentration of GHRH receptors was virtually the same regardless of tumor grade or stage. Displacement analyses demonstrated that these receptor proteins bind hGHRH and its agonistic and antagonistic analogs in a highly specific manner. Several compounds structurally related to GHRH, such as peptides of the VIP-glucagon-PACAP family, which share amino acid sequences with hGHRH or peptides unrelated to GHRH, did not inhibit the binding of radioligand JV-1-42. These tumoral GHRH receptors displayed somewhat lower binding affinity for hGHRH-(1–44)NH2, its N-terminal fragment hGHRH-(1–29)NH2, and its agonistic analog [His1,Nle27]hGHRH-(1–32)NH2 than the receptors expressed in the rat pituitary gland (13). A binding affinity at least 1 order of magnitude lower than that of JV-1-42 to tumoral GHRH receptors (13) explains why it was not possible to demonstrate GHRH-binding sites in the prostate cancer specimens examined using radiolabeled [His1,Nle27]hGHRH-(1–32)NH2, a well characterized radioligand for rat pituitary GHRH radioreceptor assays (35). This is in agreement with previous reports that no GHRH receptor binding was detected in several human experimental cancer models investigated when 125I-labeled [His1,Nle27]hGHRH-(1–32)NH2 was used as radioligand (8, 11, 13, 24, 36).
Even more important than the characterization of GHRH binding may be detection of the expression of tumoral GHRH receptor SVs in human prostate cancer specimens. Our work represents the first demonstration of the expression of SV of GHRH receptors in primary specimens of human cancer. We were able to demonstrate that SV1, which exhibits the greatest similarity to the pituitary GHRH receptors, was widely distributed, being present in 65% of the human prostate cancers. SV1 and the pituitary GHRH receptor differ only in the first three exons, encoding part of the extracellular domain of the receptor that in SV1 has been replaced by a fragment of intron 3, which has a new putative in-frame start codon (24). The presence of SV2 was shown in 12 of the 20 (60%) prostate cancer specimens. In contrast to the high incidence of SV1 and SV2, the expression of SV4 was found in only 3 samples (15%). A major part of the nucleotide sequence of SV1 (nucleotides 77–1383, encompassing exons 4–13) has more than 99% identity with the corresponding sequence of pituitary GHRH receptor cDNA (24). However, the first 334 nucleotides of both SV1 and SV2 are completely different from those in the pituitary GHRH receptors (24, 39, 40). In addition, in the 566-bp SV2 of the GHRH receptor, exon 7 is spliced out of the RNA. Thus, SV2 most likely encodes a GHRH receptor isoform truncated after the second transmembrane domain (24). The deduced protein sequences of SV1 and SV2 suggest that they possess a distinct 25-amino acid sequence at the N-terminal extracellular domain, which could serve as a signal peptide. The putative protein sequence encoded by SV1 is consistent with a functional receptor having 7 transmembrane domains, all of the intracellular loops, and a C terminal necessary for signal transduction (24, 39, 40). However, most of the large N-terminal extracellular tail, characteristic of the pituitary GHRH receptor, is deleted in SV1. This change in the structure of the GHRH receptor should result in different ligand binding affinity. The short protein sequence corresponding to SV4 lacks all transmembrane domains, implying that it is not expressed on the cell surface. A new band, less than 500 bp, has also been detected in one prostate cancer specimen after PCR amplification using primers for intron 3 and exon 8. On the basis of the size of exons, this PCR product may represent a novel 487-bp SV of GHRH receptor in prostate cancer in which exons 5 and 6 are missing, whereas exon 7 is retained. However, the sequence of this SV of GHRH receptor has not been confirmed by sequencing. Our findings on SVs of GHRH receptors are in accordance with the recent report on SVs of cholecystokinin B/gastrin receptors in pancreatic cancers (41).
That SVs of GHRH receptors play a physiopathological role in nonpituitary tissues remains to be proved. However, our recent study indicates that SV1, encoding the main isoform of GHRH receptor expressed in the NIH-3T3 mouse fibroblast cell line is involved in the proliferative responses to GHRH and its analogs (28). Thus, the expression of SV1 in the transfected cells strongly augments both the stimulatory responses to GHRH-(1–29)NH2 or GHRH agonist JI-38 and the antiproliferative effect of GHRH antagonist JV-1-38, compared with those of control cells transfected with an empty vector (28). These results suggest that GHRH receptor isoform encoded by SV1 could mediate the effect of GHRH and its antagonists on extrapituitary cells and various tumors. Various ongoing studies in our laboratory are aimed at further characterization of SVs of GHRH receptors. Thus, in addition to cross-linking of SVs of GHRH receptors with photoaffinity-labeled GHRH antagonist, we are evaluating GH, PRL, and cAMP release in response to GHRH analogs in GH3 rat pituitary tumor cells stably transfected with SV1 (Busto, R., A. V. Schally, H. Kiaris, G. Halmos, and J. L. Varga, unpublished data).
The present study also demonstrates a high incidence (86%) of mRNA for hGHRH in human prostate cancer specimens. The expression of hGHRH in primary prostate tumors is in accordance with our recent findings in LNCaP and PC-3 human androgen-independent prostate cancers (27). Chopin and Herington (26) also showed recently that the human prostate cancer lines ALVA-41, DU-145, LNCaP, and PC-3 express mRNA for GHRH and produce immunoreactive GHRH peptide in addition to expressing the mRNA encoding the SV1 isoform of GHRH receptor (26). Frohman et al. (29) were the first to report the production of GHRH in neuroendocrine pancreatic tumors. This finding was confirmed by subsequent results indicating the presence of bioactive GHRH or its gene expression in breast, endometrial, and ovarian cancers (30, 31). GHRH was also demonstrated in H-69 SCLC, and it was suggested that it may play the role of an autocrine growth factor (15). Tumoral GHRH may also be implicated in the androgen-independent progression of prostate cancer. This view is supported by our recent finding that the GHRH antagonist JV-1-38 did not influence the growth of androgen-sensitive LNCaP tumors in intact nude mice, but markedly delayed an androgen-independent progression of this prostate cancer in castrated hosts (42). In addition, studies with LNCaP androgen-dependent as well as PC-3 and DU-145 androgen-independent human prostate cancer cell lines in vitro demonstrate that GHRH stimulates, whereas various GHRH antagonists inhibit, cell proliferation (21, 36, 42). The proliferative responses of these prostate cancer cell lines to GHRH analogs were accompanied by stimulatory effects of GHRH and its agonists on the gene expression and production of prostate-specific antigen in the LNCaP line (36, 42) or IGF-II in PC-3 and DU-145 cells (21) and respective inhibitory effects of GHRH antagonists. These results suggest that GHRH acting through SV of GHRH receptors could play an important regulatory role in prostate cancer. Our results also imply that GHRH and its tumoral receptor SVs might form an autocrine mitogenic loop in prostate cancers. The antitumor effects of GHRH antagonists in prostate cancer could be exerted in part by interference with this local GHRH system.
In this study we were able to assess JV-1-42 binding in a group of 10 patients at high risk for cancer recurrence, i.e. who have locally advanced (pT3) or high grade (Gleason score 8–10) disease. Interestingly, the incidence of GHRH receptors in these patients was higher than that found in the low risk group, hinting at a possible role of GHRH and SVs of its receptors in the progression of the disease. However, the difference in incidence was not significant, as the number of specimens in each subgroup was limited. Thus, additional investigations in a large cohort of patients are required to confirm these observations.
In conclusion, our study demonstrates the expression of GHRH and SVs of GHRH receptors in surgical specimens of primary human prostate cancer. The possible significance of these findings is discussed.
Acknowledgments
We thank Dr. Rodney Davis for assistance with the collection of prostate cancer specimens, and Dr. Ana Maria Comaru-Schally for clinical support.
Footnotes
This work was supported by the Medical Research Service of the Veterans Affairs Department, an award from CaP CURE Foundation, and a grant from Zentaris AG (Frankfurt au Main, Germany) to Tulane University School of Medicine (all to A.V.S.).
Abbreviations: Bmax, Maximal binding capacity; hGHRH, human GHRH; PACAP, pituitary adenylate cyclase-activating polypeptide; SCLC, small cell lung cancer; SV, splice variant; VIP, vasoactive intestinal peptide.
Received March 5, 2002.
Accepted July 16, 2002.
References
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F. Hohla, A. V. Schally, K. Szepeshazi, J. L. Varga, S. Buchholz, F. Koster, E. Heinrich, G. Halmos, F. G. Rick, C. Kannadka, et al. Synergistic inhibition of growth of lung carcinomas by antagonists of growth hormone-releasing hormone in combination with docetaxel PNAS, September 26, 2006; 103(39): 14513 - 14518. [Abstract] [Full Text] [PDF]
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A. Havt, A. V. Schally, G. Halmos, J. L. Varga, G. L. Toller, J. E. Horvath, K. Szepeshazi, F. Koster, K. Kovitz, K. Groot, et al. The expression of the pituitary growth hormone-releasing hormone receptor and its splice variants in normal and neoplastic human tissues PNAS, November 29, 2005; 102(48): 17424 - 17429. [Abstract] [Full Text] [PDF]
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L. B. Jaeger, W. A. Banks, J. L. Varga, and A. V. Schally Antagonists of growth hormone-releasing hormone cross the blood-brain barrier: A potential applicability to treatment of brain tumors PNAS, August 30, 2005; 102(35): 12495 - 12500. [Abstract] [Full Text] [PDF]
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G. Keller, A. V. Schally, K. Groot, G. L. Toller, A. Havt, F. Koster, P. Armatis, G. Halmos, M. Zarandi, J. L. Varga, et al. Effective treatment of experimental human non-Hodgkin's lymphomas with antagonists of growth hormone-releasing hormone PNAS, July 26, 2005; 102(30): 10628 - 10633. [Abstract] [Full Text] [PDF]
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 J. B. Engel, G. Keller, A. V. Schally, G. L. Toller, K. Groot, A. Havt, P. Armatis, M. Zarandi, J. L. Varga, and G. Halmos Inhibition of Growth of Experimental Human Endometrial Cancer by an Antagonist of Growth Hormone-Releasing Hormone J. Clin. Endocrinol. Metab., June 1, 2005; 90(6): 3614 - 3621. [Abstract] [Full Text] [PDF]
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Z. Rekasi, T. Czompoly, A. V. Schally, F. Boldizsar, J. L. Varga, M. Zarandi, T. Berki, R. A. Horvath, and P. Nemeth Antagonist of growth hormone-releasing hormone induces apoptosis in LNCaP human prostate cancer cells through a Ca2+-dependent pathway PNAS, March 1, 2005; 102(9): 3435 - 3440. [Abstract] [Full Text] [PDF]
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 A. Stangelberger, A. V. Schally, J. L. Varga, M. Zarandi, K. Szepeshazi, P. Armatis, and G. Halmos Inhibitory Effect of Antagonists of Bombesin and Growth Hormone-Releasing Hormone on Orthotopic and Intraosseous Growth and Invasiveness of PC-3 Human Prostate Cancer in Nude Mice Clin. Cancer Res., January 1, 2005; 11(1): 49 - 57. [Abstract] [Full Text] [PDF]
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G. L. Toller, J. E. Horvath, A. V. Schally, G. Halmos, J. L. Varga, K. Groot, D. Chism, and M. Zarandi Development of a polyclonal antiserum for the detection of the isoforms of the receptors for human growth hormone-releasing hormone on tumors PNAS, October 19, 2004; 101(42): 15160 - 15165. [Abstract] [Full Text] [PDF]
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 Z. Szereday, A. V. Schally, J. L. Varga, C. A. Kanashiro, F. Hebert, P. Armatis, K. Groot, K. Szepeshazi, G. Halmos, and R. Busto Antagonists of Growth Hormone-Releasing Hormone Inhibit the Proliferation of Experimental Non-Small Cell Lung Carcinoma Cancer Res., November 15, 2003; 63(22): 7913 - 7919. [Abstract] [Full Text] [PDF]
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H. Kiaris, I. Chatzistamou, A. V. Schally, G. Halmos, J. L. Varga, H. Koutselini, and A. Kalofoutis Ligand-dependent and -independent effects of splice variant 1 of growth hormone-releasing hormone receptor PNAS, August 5, 2003; 100(16): 9512 - 9517. [Abstract] [Full Text] [PDF]
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 K. E. Mayo, L. J. Miller, D. Bataille, S. Dalle, B. Goke, B. Thorens, and D. J. Drucker International Union of Pharmacology. XXXV. The Glucagon Receptor Family Pharmacol. Rev., March 1, 2003; 55(1): 167 - 194. [Abstract] [Full Text] [PDF]
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M. Letsch, A. V. Schally, R. Busto, A. M. Bajo, and J. L. Varga Growth hormone-releasing hormone (GHRH) antagonists inhibit the proliferation of androgen-dependent and -independent prostate cancers PNAS, February 4, 2003; 100(3): 1250 - 1255. [Abstract] [Full Text] [PDF]
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), JV-1-38 (
), JV-1-42 (
), hGHRH-(1–44)NH2 (
), and [His1,Nle27]hGHRH-(1–32)NH2 (•). VIP (
) as well as glucagon, PACAP, JV-1-53, PG97-269, and other unrelated peptides, such as LH-releasing hormone, epidermal growth factor, somatostatin, [Tyr4]bombesin, and IGF-I, did not displace the radioligand (data not shown). One hundred percent specific binding is defined as difference between binding in the absence and that in the presence of 10-5 M JV-1-42. Each data point represents mean of at least two experiments, each performed in duplicate or triplicate. Pooled tumor membrane fractions from human prostate cancer specimens were used.
