Vasoactive intestinal peptide induces neuroendocrine differentiation in the LNCaP prostate cancer cell line through PKA, ERK, and PI3K

BACKGROUND. Neuroendocrine (NE) differentiation in prostate cancer has been correlated with unfavorable clinical outcome. The mechanisms by which prostate cancer acquires NE properties are poorly understood, but several signaling pathways have been proposed. We have previously observed that vasoactive intestinal peptide (VIP) stimulates cAMP production mainly through VPAC1 receptor, inducing NE differentiation in LNCaP cells. The aim of this study was to analyze the mechanisms involved in this process. METHODS. Reverse transcriptase (RT)-polymerase chain reaction (PCR), quantitative realtime RT-PCR, Western blotting, and immunocytochemistry were performed. RESULTS. LNCaP cells produce VIP, as demonstrated by RT-PCR and immunocytochemistry. VIP induced NE differentiation of LNCaP cells at a time as short as 1 hr of treatment, and the same occurred with the expression and secretion of neuronal-specific enolase (NSE, a NE differentiation marker). These effects were faster than those exerted by serum-deprivation. VIP induced extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation and NE differentiation by PKA-dependent and independent pathways, since the PKA inhibitor H89 partially blocked VIP-induced NE differentiation and did not affect ERK1/2 phosphorylation. mitogen-activated protein kinase kinase (MEK) and phosphoinositide 3-kinase (PI3K) appear to be also involved since the inhibitors PD98059 and wortmannin abolished ERK1/2 phosphorylation and decreased NE differentiation induced by VIP. Moreover, VIP activated Ras suggesting the involvement of a Ras-dependent pathway. CONCLUSIONS. VIP behaves as autocrine/paracrine factor in LNCaP cells by inducing NE differentiation through PKA, ERK1/2, and PI3K. Prostate 63: 44–55, 2005. # 2004 Wiley-Liss, Inc.