Abstract
Acquisition of androgen independence by prostate cancer is the key problem of prostate cancer progression. Vasoactive intestinal peptide (VIP), a neuropeptide, may act as a survival factor for prostate cancer cells under androgen deprivation. However, the molecular mechanisms by which VIP promotes the androgen-independent growth of androgen-sensitive prostate cancer cells have not been addressed. We therefore investigated the biological effect and signal pathway of VIP in LNCaP cells, a prostate cancer cell line that requires androgens for growth. We showed that low nanomolar concentrations of VIP, acting through Gs-protein-coupled VIP receptors, can induce LNCaP cell growth in the absence of androgen. Blockade of androgen-receptor (AR) in these cells by AR antagonist bicalutamide or by anti-AR small interfering RNA, inhibited the proliferative effect of VIP. In addition, VIP stimulated androgen-independent activation of AR with an EC50 of 3.0 ± 0.8 nM. We then investigated VIP-stimulated signaling events that may interact with the AR pathway in prostate cancer cells. VIP regulation of AR activation, mediated by VIP receptors, was protein kinase A (PKA)-dependent, and extracellular signal-regulated kinase 1/2 (ERK1/2) activation contributes to VIP-mediated AR activation. Furthermore, PKA-dependent Rap1 activation is required for both ERK1/2 activation and androgen-independent AR activation in LNCaP cells upon VIP stimulation. Finally, we showed that VIP-induced AR activation was also present in prostate cancer CWR22Rv1 and PC3 cells transfected with the wild-type AR. Altogether, we demonstrate that VIP acting through its Gs-protein-coupled receptors can cause androgen-independent transactivation of AR through a PKA/Rap1/ERK1/2 pathway, thus promoting androgen-independent proliferation of androgen-sensitive prostate cancer cells.
Footnotes
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This work was supported in part by National Institutes of Health grant number P20-RR018759 from the National Center for Research Resources (to Y.T., M.F.L.), CA88184 (to M.F.L.), Nebraska State LB692 (to Y.T.), and American Cancer Society RSG-07-090-01-TBE (to Y.T.).
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ABBREVIATIONS: AR, androgen receptor; ARE, androgen response element; 8-CPT-2Me-cAMP, 8-(4-chlorophenylthio)-2′-O-Me-cAMP; 8-CPT-cAMP, 8-(4-chlorophenylthio)-cAMP; N6-bnz-cAMP, N6-benzoyladenosine-cAMP; C3G, Crk SH3 domain guanine nucleotide exchanger; CRE, cAMP response element; CSS, charcoal-stripped fetal bovine serum; DHT, dihydrotestosterone; EGF, epidermal growth factor; EMSA, electrophoretic mobility shift assay; EPAC, exchange protein directly activated by cAMP; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; NEP, neutral endopeptidase; PBS, phosphate-buffered saline; PKA, cAMP-dependent protein kinase; PKI, myristoylated protein kinase inhibitor amide 14-22; PI3K, phosphoinositide-3 kinase; Rap1GAP, Rap1 GTPase-activating protein; Rap1GEF, Rap1 guanine nucleotide exchange factor; siRNA, small interfering RNA; VIP, vasoactive intestinal peptide; VPAC-R, vasoactive intestinal peptide receptor; PSA, prostate-specific antigen; HA, hemagglutinin; PKC, protein kinase C; RLU, relative luciferase activity units; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; H89, N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)-butadiene; Ly294002, Ly, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride; GF109203X, GF, 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride.
- Received January 4, 2007.
- Accepted April 12, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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