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Effects of Lysophosphatidic Acid Receptor and Free Fatty Acid Receptor Agonists on CCN1/Cyr61 Expression in Prostate Cancer Cells

Expression of CCN1/Cyr61, a member of the CCN family of matricellular proteins, can be induced by growth factors in cancer cells. CCN1 participates in cell migration and other regulatory processes in various cancers. However, the role of CCN1 in prostate cancer remains poorly understood. In previous work we observed that lysophosphatidic acid (LPA), a bioactive lipid, stimulates proliferation and migration in prostate cancer cells via the G protein‐coupled receptor (GPCR) LPAR1, and also induces CCN1 expression. We also showed that activation of free fatty acid receptor 4 (FFAR4), another GPCR, inhibits LPA‐induced proliferation and migration. For the current study, we hypothesized that CCN1 contributes to adhesion and migration of prostate cancer cells. We first confirmed that CCN1 protein levels were increased by LPA after 2‐5 hours in PC‐3 human prostate cancer cells. This response was inhibited by TUG‐891, an FFA4 agonist. When serum‐starved PC‐3 cells were treated with LPA in an extended time course from 2 minutes to 24 hours, Erk, Akt, and FAK were initially activated within 30 minutes of LPA addition, as detected by immunoblotting for the phosphorylated kinases. However, all three kinases exhibited a later phase of activation after ~6 hours. Thus, CCN1 induction is downstream of early signals initiated by LPAR1, but potentially upstream of later events. For adhesion assays, serum‐starved PC‐3 cells were incubated with and without 10 μM LPA ± 1 μM TUG‐891. Enhanced adhesion was prominent 1 hour after LPA addition, and was sustained for at least 8 hours. For migration assays, serum‐starved cells were incubated with the same agents in modified Boyden chambers. Enhanced cell migration was observed by 2 hours after LPA addition, and continued for at least 24 hours. LPA‐induced adhesion and migration were blocked by TUG‐891. In other experiments, extracellular matrix was subjected to immunoblotting and to immunofluorescence microscopy for CCN1. In the immunoblotting experiments, CCN1 protein was increased in the extracted extracellular matrix 2‐5 hours after LPA addition. In the immunohistochemistry experiments, CCN1 was detected in the extracellular space of PC‐3 cells treated with LPA for 0‐5 hours. In conclusion, the results suggest a role for LPA‐induced CCN1 in adhesion and migration in prostate cancer cells, making CCN1 a potential therapeutic target. In addition, the results further indicate that FFAR activation is a strategy to suppress both short‐ and long‐term LPA responses.