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Purpose: To study the role of the adrenomedullin system [adrenomedullin and its receptors (AMR), CLR, RAMP2, and RAMP3] in prostate cancer androgen-independent growth., Experimental Design: Androgen-dependent and -independent prostate cancer models were used to investigate the role and mechanisms of adrenomedullin in prostate cancer hormone-independent growth and tumor-associated angiogenesis and lymphangiogenesis., Results: Adrenomedullin and AMR were immunohistochemically localized in the carcinomatous epithelial compartment of prostate cancer specimens of high grade (Gleason score >7), suggesting a role of the adrenomedullin system in prostate cancer growth. We used the androgen-independent Du145 cells, for which we demonstrate that adrenomedullin stimulated cell proliferation in vitro through the cAMP/CRAF/MEK/ERK pathway. The proliferation of Du145 and PC3 cells is decreased by anti-adrenomedullin antibody (αAM), supporting the fact that adrenomedullin may function as a potent autocrine/paracrine growth factor for prostate cancer androgen-independent cells. In vivo, αAM therapy inhibits the growth of Du145 androgen-independent xenografts and interestingly of LNCaP androgen-dependent xenografts only in castrated animals, suggesting strongly that adrenomedullin might play an important role in tumor regrowth following androgen ablation. Histologic examination of αAM-treated tumors showed evidence of disruption of tumor vascularity, with depletion of vascular as well as lymphatic endothelial cells and pericytes, and increased lymphatic endothelial cell apoptosis. Importantly, αAM potently blocks tumor-associated lymphangiogenesis, but does not affect established vasculature and lymphatic vessels in normal adult mice., Conclusions: We conclude that expression of adrenomedullin upon androgen ablation in prostate cancer plays an important role in hormone-independent tumor growth and in neovascularization by supplying/amplifying signals essential for pathologic neoangiogenesis and lymphangiogenesis. Clin Cancer Res; 19(22); 6138-50. ©2013 AACR.

Clinical and experimental studies suggest that angiogenesis is a prerequisite for solid tumour growth. Glioblastoma (GBM) and pilocytic astrocytoma (PA), both angiogenic tumours display strong contrast enhancement associated with peripheral oedema in GBM but not in PA indicating differences in vascular permeability in these two types of gliomas. Here we show that expression of adrenomedullin (AM) mRNA is induced in GBM whereas is barely detectable in PA. In situ analysis of tumour specimens undergoing neovascularisation shows that the production of AM is specifically induced in a subset of GBM cells distinguished by their immediate proximity to necrotic foci (presumably hypoxic regions), suggesting a hypoxic induction of AM expression in GBM. Vascular endothelial growth factor (VEGF) mRNA levels are increased in GBM and moderate in PA. Immunohistochemical study showed that cytoplasmic AM, VEGF and HIF-1α nuclear immunoreactivity were recorded in GBM located near large necrotic areas whereas they were not expressed by PA tumour cells. Interestingly, double fluorescence immunostaining demonstrated that 85% of AM immunoreactivity colocalised with VEGF. AM transduces its effects through calcitonin receptor-like receptor/receptor activity modifying protein-2 and -3 (CLR/RAMP2 and CLR/RAMP3). Real-time quantitative RT-PCR showed expression of RAMP2, RAMP3 and CLR in PA and GBM, suggesting that AM may function as an autocrine/paracrine growth factor for GBM cells. These observations strongly support the concept that tumour angiogenesis is regulated by paracrine mechanisms and identify beside VEGF, AM as a potential tumour angiogenesis factor in vivo which constitutes a potential interesting molecular target in GBM treatment., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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 Ialpha 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 240+/-18% (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.

Adrenomedullin (AM) is a multifunctional regulatory peptide with important angiogenic and mitogenic properties. Here we identify a region of stable secondary structure in the 5′-untranslated region (5′ UTR) of human AM mRNA. Reverse transcriptase–polymerase chain reaction of the 5′ UTR consistently resulted, in addition to the product with the expected size of 155 base pair (bp), in a second product with an ∼65-bp deletion from the central region of the 5′ UTR, suggesting the presence of a secondary structure. The presence of a stem–loop structure was confirmed by probing the 5′ UTR with RNases with selectivity for single- or double-stranded RNA. We investigated the role of this stem–loop structure in expression of luciferase reporter gene in cultured cell lines. Reporter assays using a chimeric mRNA that combined luciferase and the 5′ UTR of AM mRNA demonstrated a dramatic decrease of the reporter activity owing to a decreased translation, whereas the deletion of the stem–loop structure localized between nt +31 and +95 from the cap site led to the recovery of activity. Gel migration shift assays using cytosolic extracts from mammalian cell lines demonstrate a specific binding of a cytosolic protein to riboprobes containing the 5′ UTR of AM but not to riboprobes either corresponding to other areas of the message or containing the 5′ UTR but lacking the region of secondary structure. Although we conclude that the 5′ UTR of the human AM mRNA can modulate the translation of AM mRNA in vivo, and that the predicted stem–loop structure is necessary for this inhibition, the functional consequences of the cis element-binding activity remain to be determined.Oncogene (2006) 25, 6510–6519. doi:10.1038/sj.onc.1209672; published online 22 May 2006 [ABSTRACT FROM AUTHOR]