2340 Vol. 7, 2340–2343, August 2001 Clinical Cancer Research Increased Incidence of Luteinizing Hormone-releasing Hormone Receptor Gene Messenger RNA Expression in Hormone-refractory Human Prostate Cancers Bernd Straub,1 Markus Mu¨ller, Hans Krause, to apply targeted cytotoxic LHRH analogues or monoclonal Mark Schrader, Carsten Goessl, antibodies. Ru¨diger Heicappell, and Kurt Miller Department of Urology, Universita¨tsklinikum Benjamin Franklin, INTRODUCTION Freie Universita¨t Berlin, D-12200 Berlin, Germany PC2 is the second most common cause of death among male cancer patients in Western industrialized nations (1). Me- tastases are already found at the initial diagnosis of PC in 30% ABSTRACT of cases and subsequently develop despite treatment in another There are few options for treating hormone-refractory 20%. Although organ-confined PC can be managed surgically or prostate cancer (PC). Various studies indicate that luteiniz- by radiotherapy (2), metastatic PC is initially treated by andro- ing hormone-releasing hormone (LHRH) agonists may have gen withdrawal. However, hormone-refractory tumor progres- a direct inhibitory effect on prostate tumors mediated by sion usually occurs after a few years and can then be managed specific LHRH receptors. One study evaluated LHRH re- only by palliative therapy. In the past few years, LHRH agonists ceptors in hormone-dependent PC tissue, but no data have have been increasingly used for the palliative treatment of PC. thus far been obtained on the presence of LHRH receptors Hypothalamic decapeptide LHRH plays a key role in the regu- in benign prostatic hyperplasia (BPH) and especially hor- lation of reproduction. This hormone formed in the hypothala- mone-refractory PC in patients. Thus, it is not yet clear mus stimulates the gonadotropic hormones LH and FSH in the whether LHRH receptors indicate tumor-related differenti- anterior pituitary lobe, which in turn control the gametogenic ation or even hormone-refractory dedifferentiation or are and hormonal function of the gonads. Normally, the antitumor likewise associated with BPH. The aim of this study was to effect of LHRH agonists is based on the inhibition of the determine the rate of LHRH receptor mRNA expression in pituitary-gonadal axis (3, 4). LHRH agonists and antagonists are BPH and in primary, potentially androgen-dependent and increasingly used in the treatment of advanced PCs (5–7). Var- in hormone-refractory PC with clinical progression. ious findings point to an in vitro effect on LHRH receptors Multiplex reverse transcription-PCR was used to simul- demonstrated in both androgen-sensitive and -independent PC taneously detect the expression of mRNA for LHRH recep- cell lines (8–15). In one study, the expression of mRNA for tors and ␤-actin in 48 patients with BPH, 14 with a primary, LHRH receptors was found in a larger number of human PC possibly hormone-dependent, prostate carcinoma (PPC), tissue samples. Here, both radioligand binding and RT-PCR and 18 with a hormone-refractory prostate carcinoma detection revealed (the presence of) LHRH receptors in Ͼ80% (HRPC). of the cases (16). Sixteen of 18 samples with HRPC showed intact RNA However, the above study only examined BPH tissue in a and expressed mRNA for LHRH receptors (100%). How- very small number of cases (n ϭ 3). Thus, it is not yet known ever, the RNA-intact PPC and BPH showed significantly whether the presence of LHRH receptors in PPC tissue is more lower expression of mRNA for LHRH receptors (46.2 and frequent, equal, or even reduced compared with BPH, which ,would suggest carcinoma-related differentiation. In this study .(0.0017 ؍ respectively; variance analysis: P ,55.3% The significantly higher expression of mRNA for we examined tumor samples from patients with HRPC, because, LHRH receptors in HRPC indicates that therapeutic con- to the best of our knowledge, no data are yet available on this cepts should be developed that target this site of action. In topic. Discussions have already focused on the possibility of addition to possible direct effects of LHRH agonists or tumor dedifferentiation even with an increased incidence of antagonists demonstrated previously in vitro, it seems useful mRNA for LHRH receptor expression at the stage of hormone- refractory progression. Some authors have already suggested antitumor therapy of HRPC using agonists or antagonists acting on the LHRH receptor (17). The mere detection of mRNA for Received 1/30/01; revised 5/15/01; accepted 5/23/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 2 The abbreviations used are: PC, prostate cancer; LHRH, luteinizing 1 To whom requests for reprints should be addressed, at Department of hormone-releasing hormone; BPH, benign prostatic hyperplasia; FSH, Urology, Universita¨tsklinikum Benjamin Franklin, Freie Universita¨t follicle-stimulating hormone; PPC, primary, possibly hormone-depen- Berlin, Hindenburgdamm 30, D-12200 Berlin, Germany. Phone: dent prostate carcinoma; HRPC, hormone-refractory prostate carcino- 49(30) 8445-2577; Fax: 49(30) 8445-4620; E-mail: straub@medizin. ma; TURP, transurethral resection of the prostate gland; PSA, prostate- fu-berlin.de. specific antigen; RT-PCR, reverse transcription-PCR. Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2001 American Association for Cancer Research. Clinical Cancer Research 2341 LHRH receptor expression in HRPC cell lines is not sufficient mix (10 mM each dATP, dCTP, dGTP, and dTTP), 1 ␮lof to confirm this hypothesis made by other authors. SuperScript II RNase HϪ reverse transcriptase (200 units/␮l) for The aim of this study was to detect the expression of 50 min at 42°C. Finally, the enzyme was inactivated at 65°C for mRNA for LHRH receptors in hormone-sensitive PC, HRPC, or 15 min; remaining RNA was degraded by incubation with 1 ␮l BPH tissue. of Escherichia coli RNase H (2 units/␮l) for 20 min at 37°C. For multiplex PCR, 2 ␮l of cDNA were placed into an 18-␮l reaction volume containing 2 ␮lof10ϫ PCR buffer (see MATERIALS AND METHODS ␮ ␮ above), 1.2 lof25mM MgCl2, 0.4 lof10mM deoxynucle- Tissue Samples. Thirty-two PC tissues and 48 BPH otide triphosphate mix (see above), and 0.2 ␮l of Taq DNA samples were collected from patients who underwent surgery in polymerase (Roche, Mannheim, Germany). Primers for LHRH our department. Examinations were conducted in agreement receptor were: sense, 5Ј-GAC CTT GTC TGG AAA GAT with the Helsinki Declaration. Institutional Review Board ap- CC-3Ј; antisense, 5Ј-CAG GCT GAT CAC CAC CAT CA-3Ј proval was obtained for this study. Each patient signed a consent (18); and for ␤-actin: sense, 5Ј-GTG GGG CGC CCC AGG form approved by the Committee on Human Rights in Research CAC CA-3Ј; and antisense, 5Ј-CTC CTT AAT GTC ACG CAC of our institution. GAT TTC-3Ј. Thirty-five cycles (94°C for 1 min, 55°C for 1 Fourteen tissue samples were taken from patients with min, and 72°C for 1 min, followed by a final extension at 72°C primary, untreated PC (patient ages, 54–73 years; mean age, for 7 min) were run on a 9600 thermal cycler (Applied Biosys- 62.1 Ϯ 1.7) during radical prostatectomy in the following man- tems, Ueberlingen, Germany) for LHRH receptors and ␤-actin. ner. Immediately after removing the gland from the operation Negative controls were concomitantly run to confirm that the site, a 7–9-mm-thick slice was cut out of the sample in the area samples were not cross-contaminated. A sample with 1 ␮lof of the positive punch biopsy, divided into 12 blocks of approx- diethylpyrocarbonate-treated water, instead of reverse tran- imately the same size, and stored at Ϫ80°C; the remainder was scriptase, was concomitantly examined for each of the reaction put in formalin for conventional histology. Only those samples units described above. Two control assays were performed from containing almost exclusively, i.e., a minimum of Ͼ90%, pure each tissue. Subsequently, the PCR products were subjected to carcinoma tissue were included for further processing. With this gel electrophoresis in 1.5% agarose gels containing 0.5 ␮g/ml method, the 12 blocks from each of the 42 different biopsy ethidium bromide. Variables were examined for Gaussian dis- specimens had to be processed to obtain 14 different samples tribution and by ANOVA, and the results were given as mean Ϯ that fulfilled the above-mentioned criteria. Five 30-␮m-thick SE. The significance of differences (P Յ 0.05) between the slices were cut from the sample blocks in the cryostat (Kryostat three groups was assessed by the least squared differences test 2800; Leica Instruments, Nussloch, Germany) and submitted to using the Statistical Package from STATISTICA (StatSoft, further processing (see below). We applied the above proce- Tulsa, OK). dures, because the RNA quality of the tumor material obtained by laser microdissection was not adequate for RT-PCR because RESULTS of extensive sample processing. Thirteen of 14 PPC samples (93%) expressed the “house- Tissue slices from 42 BPH patients (patient ages, 48–87 keeping gene” ␤-actin as proof of RNA integrity, and 6 ex- years; mean age, 67.8 Ϯ 1.4), who underwent TURP, were pressed (46.2%) mRNA for LHRH receptors, although there initially stored at Ϫ80°C. These frozen tissue slice blocks were was no statistically significant correlation between the presence subsequently prepared with the cryostat as described above. of mRNA for LHRH receptors and the Gleason score (mean, Tissue slices from 18 patients with a HRPC (patient ages, 6.9 Ϯ 0.5), pathological staging or grading, the preoperative 57–88 years; mean age, 72.1 Ϯ 2.4), who underwent palliative PSA value, or age (Table 1).