Oncogene (2012) 31, 2075–2089 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE Lysophosphatidic acid and sphingosine-1-phosphate promote morphogenesis and block invasion of prostate cancer cells in three-dimensional organotypic models VHa¨rma¨1, M Knuuttila2, J Virtanen1,2, T Mirtti3,4, P Kohonen2, P Kovanen4, A Happonen5, S Kaewphan6, I Ahonen2, O Kallioniemi3, R Grafstro¨m1,7,JLo¨tjo¨nen8 and M Nees1 1Medical Biotechnology Knowledge Centre, VTT Technical Research Centre of Finland, Turku, Finland; 2Biotechnology Centre, University of Turku, Turku, Finland; 3Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland; 4Department of Pathology, Haartman Institute, University of Helsinki and HUSLAB, Helsinki University Central Hospital, Helsinki, Finland; 5Department of Signal Processing, Tampere University of Technology, Tampere, Finland; 6Department of Information Technology, University of Turku, Turku, Finland; 7Laboratory for Toxicology, Karolinska Institute, Stockholm, Sweden and 8Knowledge Intensive Services, VTT Technical Research Centre of Finland, Tampere, Finland Normal prostate and some malignant prostate cancer LPAR1 and Ga12/13 signalling, regulating cell motility (PrCa) cell lines undergo acinar differentiation and form and invasion versus epithelial maturation. spheroids in three-dimensional (3-D) organotypic culture. Oncogene (2012) 31, 2075–2089; doi:10.1038/onc.2011.396; Acini formed by PC-3 and PC-3M, less pronounced also published online 26 September 2011 in other PrCa cell lines, spontaneously undergo an invasive switch, leading to the disintegration of epithelial Keywords: prostate cancer; epithelial plasticity; structures and the basal lamina, and formation of bioactive lipids; G-protein coupled receptors; lysopho- invadopodia. This demonstrates the highly dynamic nature sphatidic acid; sphingosine-1-phosphate of epithelial plasticity, balancing epithelial-to-mesenchy- mal transition against metastable acinar differentiation. This study assessed the role of lipid metabolites on epithelial maturation. PC-3 cells completely failed to form Introduction acinar structures in delipidated serum. Adding back lysophosphatidic acid (LPA) and sphingosine-1-phosphate The mechanisms promoting local invasion and metas- (S1P) rescued acinar morphogenesis and repressed inva- tasis of castration-resistant, late stage prostate cancer sion effectively. Blocking LPA receptor 1 (LPAR1) (PrCa) are incompletely understood and poorly recapi- functions by siRNA (small interference RNA) or the tulated by standard 2-dimensional (2-D) monolayer cell specific LPAR1 inhibitor Ki16425 promoted invasion, culture and invasion models (for example, transwell while silencing of other G-protein-coupled receptors migration/Boyden chambers and scratch-wound assays). responsive to LPA or S1P mainly caused growth arrest 2-D models fail to support the formation of multi- or had no effects. The G-proteins Ga12/13 and Gai were cellular structures and epithelial barriers such as the identified as key mediators of LPA signalling via basement membrane (BM). In contrast, advanced model stimulation of RhoA and Rho kinases ROCK1 and 2, systems in three-dimensional (3-D) tumor microenvir- activating Rac1, while inhibition of adenylate cyclase and onment promote the formation of organotypic struc- accumulation of cAMP may be secondary. Interfering tures with relevant cell–cell and cell–matrix interactions with these pathways specifically impeded epithelial (Brekhman and Neufeld, 2009), epithelial polarization polarization in transformed cells. In contrast, blocking and differentiation. In particular, models that utilize the same pathways in non-transformed, normal cells physiologically relevant ECM such as collagens or promoted differentiation. We conclude that LPA and laminin (Matrigel) increasingly gain relevance (Reuter LPAR1 effectively promote epithelial maturation and et al., 2009; Inman and Bissell, 2010; Ridky et al., 2010). block invasion of PrCa cells in 3-D culture. The analysis 3-D cultures represent an opportunity to investigate of clinical transcriptome data confirmed reduced expres- dynamic morphogenetic processes like epithelial-to- sion of LPAR1 in a subset of PrCa’s. Our study mesenchymal transition (Chu et al., 2009), a central demonstrates a metastasis-suppressor function for mechanism determining tumor cell motility, invasiveness and drug resistance (Mani et al., 2008; Kalluri and Weinberg, 2009). We have recently reported a miniatur- Correspondence: Dr M Nees, Medical Biotechnology Knowledge ized 3-D platform to analyze the morphology of PrCa Centre, VTT Technical Research Centre of Finland, Varsinais Suomi, cell lines in laminin-rich ECM (lrECM) (Harma et al., PO Box 106, Turku FI 20520, Finland. E-mail: matthias.nees@vtt.fi 2010). Malignant PC-3 and PC-3M cells initially show Received 5 March 2011; revised 26 June 2011; accepted 28 July 2011; normal acinar differentiation, but later undergo sponta- published online 26 September 2011 neous transformation into aggressive stellate structures Lysophosphatidic acid and sphingosine-1-phosphate versus their functions VHa¨rma¨ et al 2076 (‘invasive switch’). After the initial formation of automated image analysis (ACCA). At high density invadopodia, followed by the disintegration of symme- (2500 cells/well), invasion commences preferentially in trical spheroids and BM degradation, malignant cells dense areas at earlier time points. Adding fresh medium invade the lrECM as multicellular strings, concomitant to invasive 3-D cultures results in temporary retraction with the re-organization of cell–cell and cell–matrix of invasive processes. contacts, and altered integrin cell adhesion signalling. Next, medium was supplemented with increasing Lysophosphatidic acid (LPA) and sphingosine-1- quantities of fetal bovine serum (FBS). In the absence phosphate (S1P) are simple, water soluble bioactive of FBS, cells failed to form round spheroids, and lipids that regulate diverse cellular functions like cell- invaded the lrECM from day 1 (Figure 1b). FBS proliferation (Gibbs et al., 2009), differentiation (Kim concentrations 42.5% supported acinar morphogenesis et al., 2002; Bagga et al., 2004), apoptosis (Goetzl et al., and suppressed invasive behavior, indicating that 1999b; Deng et al., 2002), migration (Shida et al., 2003; differentiation-promoting factors were sufficiently sup- Hao et al., 2007; Kim et al., 2008; Wang et al., 2008; plied by 2.5% FBS. In contrast, cultures supplemented Li et al., 2009a, b) and adhesion (Sawada et al., 2002; with charcoal-delipidated serum (CSS), removing the Smicun et al., 2007; Devine et al., 2008) in many cell most lipophilic components including LPA and S1P types. Physiological LPA levels between 0.1-25 mM are (Lee et al., 1998a, 1998b), failed to develop spheroids found in serum (Tigyi and Miledi, 1992), ascitic even at 20% FBS (Figure 1c). This indicates repression effusions (Westermann et al., 1998) and inflammatory of invasive properties by lipophilic factors. fluids (Fourcade et al., 1995). Large amounts of LPA and S1P are secreted by platelets (Eichholtz et al., 1993), The bioactive lipids LPA and S1P promote epithelial adipocytes (Valet et al., 1998) and fibroblasts (Fukami maturation and suppress invasive transformation and Takenawa, 1992; Olivera and Spiegel, 1993). LPA To identify the postulated lipophilic regulators, we tested a and S1P mediate intracellular actions mainly via G- panel of steroids, growth factors and vitamins (Supplemen- protein-coupled receptors, encoded by the LPA-recep- tary Figure S1). Re-constitution of androgens, estrogens, tors LPAR1-6 and S1P receptors S1PR1-5, and coupled progesterone, glucocorticoids, vitamin A (retinoic acid) to stimulatory or inhibitory G-proteins, including Ga , i/o or D (cholecalciferol) failed to support acinar differ- Ga ,Ga and Ga . Preferences for certain G-protein 3 s q 12/13 entiation. Next, we investigated lipids found at signi- utilization depend on the receptors, cell environment, ficant concentrations in serum, plasma and lymphatic differentiation and adaptation to acute stress/signalling fluids (prostaglandins, eicosanoids and thromboxanes). conditions. LPA and S1P receptors trigger intracellular Only addition of LPA and S1P effectively suppressed signalling cascades such as Ca2 þ mobilization (Meyer zu invasion (Figure 2), maintaining differentiation, polari- Heringdorf et al., 1998), activation of the Rho-GTPases, zation and complete BM. LPA was less potent than S1P, accumulation of cytoplasmic cAMP and actin rearran- with strong invasion-suppressive effects for 36 h and gement, resulting in dramatic changes of cell morphology 42 h at 1.0 and 10 mM, respectively (Figure 2a). With and motility (van Dijk et al., 1998). LPA and S1P are S1P, effects lasted for 4100 h (Figure 2b). In delipidated associated with the progression of malignancies including medium (Figure 2c, CSS), the BM started to disintegrate ovarian (Hong et al., 1999; Fang et al., 2002), breast after 24 h and was completely disrupted after 72-96 h (Goetzl et al., 1999a; Nava et al., 2002) and colorectal (compare also Figure 6). Both LPA and S1P potently cancers (Shida et al., 2004; Kawamori et al., 2006). repressed BM degradation and invasion. However, high However, their functions and relevance in PrCa progres- concentrations of S1P, but not LPA, caused morpho- sion, invasion and motility remain controversial (Gibbs logical defects and disorganization