Roles for CCN proteins in Lysophosphatidic Acid and Sphingosine 1‐Phosphate Signaling in Prostate Cancer Cells

Abstract

Introduction Cysteine rich angiogenic factor (CCN1/Cyr61) and connective tissue growth factor (CCN2/CTGF) are members of the CCN family of matricellular proteins. These secreted proteins interact with extracellular matrix proteins and integrins to modulate cell signaling. CCN proteins have been implicated in survival and other mitogenic responses in various cancers. CCN1 and CCN2 are the two early inducible genes in this family. Lysophosphatidic acid (LPA) and sphingosine‐1‐phosphate (S1P) are lipid growth factors known to contribute to tumor progression in prostate cancer. In previous work, our group showed that LPA stimulates proliferation and migration in prostate cancer cells via the G protein‐coupled receptor LPAR1. Free fatty acid receptor 4 (FFAR4) agonists, inhibit LPA‐induced responses in prostate cancer cells. We also demonstrated that CCN1 expression is induced by LPA in human prostate cancer cells. However, the roles of CCN1 and CCN2 in cancer cell signaling have not been fully elucidated. Hypothesis We hypothesize that CCN proteins contribute to LPA‐ and S1P‐induced adhesion and mitogenic signaling in prostate cancer cells. Methods and Results PC‐3 human prostate cancer cells were used in the current study. For all experiments, cells were serum starved for 24 hours and then treated with and without 10 µM LPA or S1P. Immunoblotting showed that CCN1 and CCN2 were induced by LPA after 2‐5 hours in PC‐3 cells. The response was inhibited by the LPA receptor antagonist Ki1625. TUG‐891, an FFAR4 agonist, inhibited LPA‐induced CCN1 expression in PC‐3 cells. S1P also induced CCN1 expression in PC‐3 cells. In related experiments, whole cell extracts, and extracellular matrix were subjected to immunoblotting for CCN1, and immunofluorescence microscopy was used to localize the induced CCN1. CCN1 protein levels in extracellular matrix were increased 2‐5 hours after LPA treatment, as compared to untreated controls, and CCN1 was detected outside the cells by confocal microscopy. In cell adhesion assays, LPA‐treated PC‐3 cells exhibited an increase in adhesion to fibronectin‐coated 96‐well plates that was detected by 2 hours. Erk MAPK activation in response to LPA was biphasic, with the later phase occurring after CCN1/2 induction. Conclusion The results of this study suggest that CCN proteins may play roles in LPA‐ and S1P‐induced signaling in prostate cancer cells and are thus potential therapeutic targets.

Global Proteomics Analysis of Lysophosphatidic Acid Signaling in PC-3 Human Prostate Cancer Cells: Role of CCN1

“…This is in agreement with our previous results demonstrating prominent induction of CCN1 by LPA in PC-3 cells [17]. The 3 h time point used for the LPA incubation was within the peak time of CCN1 induction and was approximately 1 h after maximal induction as previously detected by immunoblotting [17]. Among the top 20 proteins significantly up-regulated by LPA were metastasis associated in colon cancer protein 1 (MACC1), thrombospondin-1 (TSP1), transcription factor Jun-B, and Dickkopf-related protein 1 (DKK1).…”

“…Notably, CCN1 was one of the most highly up-regulated proteins in the LPA treatment group. This is in agreement with our previous results demonstrating prominent induction of CCN1 by LPA in PC-3 cells [17]. The 3 h time point used for the LPA incubation was within the peak time of CCN1 induction and was approximately 1 h after maximal induction as previously detected by immunoblotting [17].…”

“…Our studies have shown that CCN1 is detected in human prostate cancer cells 2-6 h after the addition of LPA [5,17]. This led us to hypothesize that CCN1 plays a role in the intermediate phase of LPA response in these cells.…”

“…This led us to hypothesize that CCN1 plays a role in the intermediate phase of LPA response in these cells. Our previous results show that, in PC-3 cells, CCN1 plays an important role in LPA-induced cell adhesion that is observed after two hours [17]. Others have shown that CCN1 is required for PC-3 cell proliferation [20].…”

“…CCN1 is induced through the activation of the transcriptional co-activator YAP in cancer cells [16]. Induced CCN1 is rapidly secreted from cells, appearing in the extracellular matrix [17]. Since CCN1 is a ligand for integrins, it is likely that further downstream signaling is mediated, at least in part, through integrins [18,19].…”