Lysophosphatidic acid and sphingosine-1-phosphate promote morphogenesis and block invasion of prostate cancer cells in three-dimensional organotypic models

Normal prostate and some malignant prostate cancer (PrCa) cell lines undergo acinar differentiation and form spheroids in three-dimensional (3-D) organotypic culture. Acini formed by PC-3 and PC-3M, less pronounced also in other PrCa cell lines, spontaneously undergo an invasive switch, leading to the disintegration of epithelial structures and the basal lamina, and formation of invadopodia. This demonstrates the highly dynamic nature of epithelial plasticity, balancing epithelial-to-mesenchymal transition against metastable acinar differentiation. This study assessed the role of lipid metabolites on epithelial maturation. PC-3 cells completely failed to form acinar structures in delipidated serum. Adding back lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) rescued acinar morphogenesis and repressed invasion effectively. Blocking LPA receptor 1 (LPAR1) functions by siRNA (small interference RNA) or the specific LPAR1 inhibitor Ki16425 promoted invasion, while silencing of other G-protein-coupled receptors responsive to LPA or S1P mainly caused growth arrest or had no effects. The G-proteins Gα12/13 and Gαi were identified as key mediators of LPA signalling via stimulation of RhoA and Rho kinases ROCK1 and 2, activating Rac1, while inhibition of adenylate cyclase and accumulation of cAMP may be secondary. Interfering with these pathways specifically impeded epithelial polarization in transformed cells. In contrast, blocking the same pathways in non-transformed, normal cells promoted differentiation. We conclude that LPA and LPAR1 effectively promote epithelial maturation and block invasion of PrCa cells in 3-D culture. The analysis of clinical transcriptome data confirmed reduced expression of LPAR1 in a subset of PrCa's. Our study demonstrates a metastasis-suppressor function for LPAR1 and Gα12/13 signalling, regulating cell motility and invasion versus epithelial maturation

Extracellular loop 3 of the noradrenaline transporter contributes to substrate and inhibitor selectivity

The human noradrenaline transporter (NET) and 5-hydroxytryptamine (5-HT) transporter (SERT) are inhibited by antidepressants and psychoactive drugs such as cocaine. Both substrates and inhibitors bind in the transmembrane core of the protein, but molecular divergence at the binding site is not sufficient to account for the NET-selective and SERT-selective inhibition of the antidepressants, desipramine and citalopram, respectively. We considered that the poorly conserved third extracellular loop may contribute to these differences. We substituted single amino acid residues of the third extracellular loop in NET for equivalents from SERT, transiently transfected COS-7 cells with WT NET, 13 mutant NETs and WT SERT, and measured [ 3H]noradrenaline uptake, [3H]nisoxetine binding and [ 3H]5-HT uptake. Mutants F299W, Y300Q, R301K and K303L, at the C-terminal end of EL3, all showed significantly decreased [3H] nisoxetine binding, indicative of a reduced cell surface expression. Most mutants differed little, if at all, from WT NET regarding [3H] noradrenaline uptake; however, the I297P mutant showed no significant uptake activity despite intact cell surface expression, and the A293F mutant showed a significantly slower transporter turnover than WT NET in addition to [ 3H]5-HT uptake that was significantly greater than that of WT NET. The A293F mutation also decreased desipramine potency and increased the inhibition of [3H]noradrenaline uptake by citalopram compared to WT NET. These results suggest that the third extracellular loop allosterically regulates the ability of the transmembrane domains to transport substrates and bind inhibitors and thus contributes to the selectivity of substrates and antidepressants for NET and SERT