1. ¹Inserm E 359, Marseille, France
2. ²Université de la Méditerranée Aix-Marseille II, Faculté de Médecine Secteur Nord, IFR Jean Roche, Laboratoire de Cancérologie Expérimentale, Bd Pierre Dramard, Marseille, France
3. ³Université de la Méditerranée Aix-Marseille II, Faculté de Médecine, Laboratoire de l'Adhésion et de la Signalisation EA3281, Marseille, France
4. ⁴Assistance Publique Hôpitaux de Marseille, CHU Timone, Service Radiothérapie, Marseille, France
5. ⁵Inserm UMR 626, Marseille, France
6. ⁶Assistance Publique Hôpitaux de Marseille, CHU Nord, Service Urologie, Marseille, France
7. ⁷CNRS FRE 2738, Marseille, France
8. ⁸Assistance Publique Hôpitaux de Marseille, CHU Nord, Laboratoire de Transfert en Oncologie Biologique et Moléculaire, Marseille, France

Correspondence: Dr L'Houcine Ouafik, Université de la Méditerranée Aix-Marseille II, Faculté de Médecine Secteur Nord, IFR Jean Roche, Laboratoire de Cancérologie Expérimentale, Bd Pierre Dramard, Marseille cedex 20, 13916, France. E-mail: lhoucine.ouafik@univmed.fr

⁹These authors contributed equally to this work.

Received 21 July 2006; Revised 4 June 2007; Accepted 6 June 2007; Published online 16 July 2007.

Abstract

Neuroendocrine (NE) differentiation in prostate cancer (CaP) has been reported to be an early marker associated with the development of androgen independence. The mechanisms by which CaP acquires NE properties are poorly understood. In this study, a putative role of adrenomedullin (AM) in the NE differentiation was investigated. The expression of AM and AM receptors (calcitonin receptor-like receptor (CRLR)/receptor activity modifying protein-2 and -3 (RAMP2 and RAMP3) was evaluated after experimental manipulation of androgen status. Levels of AM mRNA and immunoreactive AM (ir-AM) increased four- to sevenfold in androgen-sensitive LNCaP cells after androgen withdrawal in vitro and in LNCaP xenografts in animals after castration. Treatment of LNCaP cells with androgen analogue (dihydrotestosterone; 10^-9 M) prevented the increase in AM mRNA and ir-AM levels. Interestingly, the expression of CRLR, RAMP2 and RAMP3 is not regulated by androgen status. We demonstrate that in the presence of serum, AM is able to induce an NE phenotype in LNCaP cells via CRLR/RAMP2 and RAMP3, which includes extension of neuritic processes and expression of the neuron-specific enolase (NSE), producing cGMP in a dose-dependent manner, which is mediated by a pertussis toxin-sensitive GTP-binding protein. 8-bromo-cGMP mimicked the effects of AM on cell differentiation. We demonstrate that AM induces a G-kinase I translocation to the nucleus. The protein kinase G inhibitor KT-5823 inhibited the neurite outgrowth induced by both AM and 8-bromo-cGMP. In noncastrated animals, administration of AM enhanced expression of NSE and chromogranin A in LNCaP xenografts with a significant increase of NSE levels in serum and no changes in tumor growth. In castrated animals, intraperitoneal injection of AM resulted in a 24018% (P<0.001) increase in tumor volume 36 days after treatment, indicating that the nature of effect of AM in CaP depends on the presence or absence of endogenous androgen. Together, these results demonstrate that AM may function as a mediator of NE-like differentiation in culture as well as in vivo and indicate that its production may be important for tumor resurgence following androgen ablation.