[CANCER RESEARCH 61, 1196 –1206, February 1, 2001] Expression of Adrenomedullin and Peptide Amidation Activity in Human Prostate Cancer and in Human Prostate Cancer Cell Lines 1 Palma Rocchi, Franc ¸oise Boudouresque, Alfredo J. Zamora, Xavier Muracciole, Eric Lechevallier, Pierre-Marie Martin, and L’Houcine Ouafik 2 Laboratoire de Cance ´rologie Expe ´rimentale-EA 2671/Laboratoire de Transfert, Assistance Publique-Hopitaux de Marseille (AP-HM), 13916 Marseille Cedex 20 [P. R., F. B., X. M., P-M. M., L. O.]; Interactions Cellulaires Neuroendocriniennes Unite ´ Mixte de Recherche 6544 Centre National de Recherche Scientifique, Institute Federatif de Recherche Jean Roche, Faculte ´ de Me ´decine Nord 13916 Marseille Cedex 20 [A. J. Z.]; Service de Radiothe ´rapie, Centre Hospitalier Universitaire Timone, Marseille 13005 AP-HM [X. M.]; and Service d’Urologie, Hopital Salvator AP-HM Marseille 13009 [E. L.], France ABSTRACT After therapeutic hormone deprivation, prostate cancer (CaP) cells often develop androgen-independent growth through not-well-defined mechanisms. The presence of neuroendocrine (NE) cells is often greater in prostate carcinoma than in normal prostate, and the frequency of NE cells correlates with tumor malignancy, loss of androgen sensitivity, increase of autocrine-paracrine activity, and poor prognosis. In some CaPs, neu- ropeptides have been previously implicated as growth factors. Peptidyl- glycine -amidating monooxygenase (PAM) is the enzyme producing -amidated bioactive peptides from their inactive glycine-extended pre- cursors. In the present work, we demonstrate that androgen-independent PC-3 and DU145 cell lines, derived from human CaP, express PAM in vitro and in xenografts implanted in athymic nude mice, indicating that they are able to produce -amidated peptides. Contrarily, barely detect- able levels of PAM were found in the androgen-sensitive LNCaP cell line. We also show that whereas PC-3 and DU145 cells produce and secrete adrenomedullin (AM), a multifunctional amidated peptide, no expression was found in LNCaP cells. We further demonstrate that AM acts as a growth factor for DU145 cells, which suggests the existence of an autocrine loop mechanism that could potentially drive neoplastic growth. PAM mRNA levels were found to be 3-fold higher in prostate adenocarcinomas compared with that of human benign prostate hyperplasia (BPH) as demonstrated by real-time quantitative reverse transcription-PCR. The analysis of AM message expression in BPH and CaP (Gleason’s score, 6 –9) shows a clear distinction between benign and CaP. The expression was detected only in adenocarcinomas tissues with a marked increase in samples with a high Gleason’s score. Immunocytochemically, AM was localized in the carcinomatous epithelial compartment. NE phenotype, assessed after the immunocytochemical localization of neuron-specific enolase (NSE), was found in both the epithelial and the stromal compart- ments of cancers; in BPH, only some spare basal cells were NSE-labeled. Cancer progression could be accelerated by peptides secreted by a popu- lation of cells capable of inducing androgen-independent tumoral growth via autocrine-paracrine mechanisms. INTRODUCTION CaP 3 is currently the second leading cause of cancer death in men (1). Because androgens stimulate tumoral growth, hormone depriva- tion represents at present the main treatment of advanced CaP. Pros- tate is a tubuloalveolar gland that contains a simple, slowly renewing epithelium composed of three cell types, namely secretory, basal, and NE cells (2). Secretory (luminal) cells predominate. Basal cells are lodged beside the basement membrane and constitute the stem cell population in mature glands (3, 4). NE cells are rare; scattered among the acini and ducts (5, 6) they secrete a variety of factors controlling gland development and maintenance (7–9). CaP often displays focal NE phenotype (largely named NE differ- entiation). Clusters of NE cells have been reported to be a constituent of most prostate adenocarcinomas (10), with incidences rising even to 100% (11). Recent studies suggest that the presence of this phenotype is associated with the androgen-independent progression of the cancer (12, 13), which renders prognosis quite unfavorable (13, 14). In this context, it is noteworthy that human NE cells do not express detect- able levels of androgen receptors (15). It has also been shown that androgen withdrawal leads to drastic and chronic reduction in prostate blood flow (16, 17), decreased cell proliferation and androgen-depen- dent angiogenesis, and increased apoptosis of androgen-dependent CaP (18). It seems, therefore, plausible to postulate that androgen depletion could inhibit the activity of autocrine-paracrine growth factors engaged in cell proliferation, and, consequently, the evolution of androgen-insensitive CaP has necessarily to involve the generation or activation of alternative androgen-independent growth pathways. Recently, a variety of neuropeptides have been shown in CaP secreted by foci of NE cells (11). On the other hand, many important peptides, such as substance P, neuropeptide Y, vasoactive intestinal peptide, galanin, gastrin-releasing peptide, and thyrotropin-releasing hormone (19, 20), are all -amidated at their COOH terminus, a structural modification that is essential for their biological activity (20). Amidation represents an important step in the maturation of as many as one-half of known peptidic hormones and growth factors (20). The only enzyme complex catalyzing this key posttranslational modification has been identified as the PAM (EC 1.14.17.3), which consists of two enzymes acting sequentially to convert peptidylgly- cine substrates into -amidated products and glyoxylate. The first enzymatic step is carried out by the PHM, which, in the presence of ascorbate, copper, and molecular oxygen, produces an -hydroxylated intermediary product. The subsequent step is then performed by the PAL, which catalyzes the synthesis of the final -amidated peptide and glyoxylate (20). Several human PAM cDNAs have been cloned (21). We have localized the human PAM gene, whose primary tran- script is subject to alternative splicing, on chromosome 5q14 –5q21 (22, 23). PAM proteins undergo tissue-specific endoproteolytic cleav- age, yielding both soluble and membrane-associated PHM and PAL (24). The consistency in CaP of both NE cells and neuropeptide synthesis prompted us to investigate the interrelations between NE phenotype and PAM expression. The presence of PAM should indicate the active synthesis of -amidated peptides functioning as growth factors on tumoral cells. In the present work, the analysis performed on cell lines derived from human prostate carcinoma, either cultured or xe- Received 3/13/00; accepted 11/20/00. 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. 1 Supported by l’Association pour la Recherche sur les Tumeurs de la Prostate (ARTP) and the Assistance Publique-Hopitaux de Marseille (AP-HM). L. O. and A. J. Z. are Institut National de la Sante ´ et de la Recherche Me ´dicale investigators. The Conseil Regional PACA and Ipsen-Biotech financially supported P. R. 2 To whom requests for reprints should be addressed, at Laboratoire de Cance ´rologie Expe ´rimentale-EA 2671/Laboratoire de Transfert, Assistance Publique-Hopitaux de Mar- seille, France. Phone: 33-491-698-882; Fax: 33-491-090-171; E-mail: ouafik.h@jean- roche.univ-mrs.fr. 3 The abbreviations used are: CaP, cancer of the prostate; NE, neuroendocrine; PAM, peptidylglycine -amidating monooxygenase; PHM, peptidylglycine -hydroxylating monooxygenase; PAL, peptidylglycine -hydroxyglycine -amidating lyase; AM, ad- renomedullin; RT-PCR, reverse transcription-PCR; BPH, benign prostate hyperplasia; NSE, neuron-specific enolase; IR-AM, immunoreactive AM; AP-HM, Assistance Pub- lique-Hopitaux de Marseille; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ADK, adenocarcinoma. 1196 Research. on February 16, 2016. © 2001 American Association for Cancer cancerres.aacrjournals.org Downloaded from