Adrenomedullin, an autocrine/paracrine factor induced by androgen withdrawal, stimulates ‘neuroendocrine phenotype’ in LNCaP prostate tumor cells

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 À9 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 Ia translocation to the nucleus. The protein kinase G inhibitor KT-5823 inhibited the neurite outgrowth induced by both AM and 8-bromocGMP. 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 240718% (Po0.001) 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 NElike differentiation in culture as well as in vivo and indicate that its production may be important for tumor resurgence following androgen ablation.

A gene signature identified using a mouse model of androgen receptor‐dependent prostate cancer predicts biochemical relapse in human disease

“…ADM has been shown to promote prostate cancer cell growth 30,38 and was the only gene in this group with multiple probes on the microarray, both of which showed concordant upregulation of gene expression (3.63 and 3.64-fold) in AR-E231G mice. CITED1 is a putative co-activator of AR 39 and was selected for functional characterization, because it was the most highly upregulated, potentially novel prostate cancer gene with a human homolog.…”

“…Adm and Cited1 were two genes found to be differentially expressed in the prostates of AR-E231G mice compared to control mice at 12 weeks of age. ADM is an angiogenic factor that is induced in the absence of androgens, 30,38 promotes prostate cancer cell growth in vitro and in vivo 30 and is required for maintenance of the neuroendocrine phenotype in LNCaP cells. 30 CITED1 is a poorly characterized transcriptional coactivator that may enhance the transcriptional activity of the AR.…”

“…ADM is an angiogenic factor that is induced in the absence of androgens, 30,38 promotes prostate cancer cell growth in vitro and in vivo 30 and is required for maintenance of the neuroendocrine phenotype in LNCaP cells. 30 CITED1 is a poorly characterized transcriptional coactivator that may enhance the transcriptional activity of the AR. 39 The levels of these factors were elevated in AR-E231G mice, and both were shown to be required for normal viability and proliferation in the LNCaP cell line.…”

“…Using Robust Multiarray Averaging, 132 genes (74 increased, 58 decreased) were differentially expressed between the two mouse strains (Supporting Information Tables 3 and 4). Sixteen of these genes (12.1%) have previously been reported to be regulated by androgens (Supporting Information Table 5), including Adm and Greb1, 30,31 suggesting that AR-E231G and AR-T857A may possess differential transcriptional activity at certain ARtargeted loci. Notable genes elevated in the AR-E231G model included Rb1 32 and Ccr2, 33 both of which have been shown to be overexpressed in clinical prostate cancer, and Pmaip1, a gene that is predictive of BCR in human disease.…”

Adrenomedullin and calcitonin gene-related peptide receptors in endocrine-related cancers: opportunities and challenges

“…AM can act directly to stimulate cell growth and inhibit apoptosis in a variety of tumour cells (Forneris et al 2001, Martinez et al 2002, Albertin et al 2005. AM can change the phenotype of cells, leading them to exhibit tumourigenic behaviour, for example, in prostate cancer, AM promotes the appearance of a neuroendocrine phenotype in cells (Martinez et al 2002, Berenguer et al 2008. AM may promote tumour blood and lymphatic angiogenesis, providing a supply of nutrients and oxygen to the tumour but also providing a means for tumour cells to spread (Garayoa et al 2000, Fritz-Six et al 2008, Ichikawa-Shindo et al 2008.…”