Lysophosphatidic acid (LPA) refers to a family of simple phospholipids that act as ligands for G protein-coupled receptors. While LPA exerts effects throughout the body in normal physiological circumstances, its pathological role in cancer is of great interest from a therapeutic viewpoint. The numerous LPA receptors (LPARs) are coupled to a variety of G proteins, and more than one LPAR is typically expressed on any given cell. While the individual receptors signal through conventional GPCR pathways, LPA is particularly efficacious in stimulating cancer cell proliferation and migration. This review addresses the mechanistic aspects underlying these pro-tumorigenic effects. We provide examples of LPA signaling responses in various types of cancers, with an emphasis on those where roles have been identified for specific LPARs. While providing an overview of LPAR signaling, these examples also reveal gaps in our knowledge regarding the mechanisms of LPA action at the receptor level. The current understanding of the LPAR structure and the roles of LPAR interactions with other receptors are discussed. Overall, LPARs provide insight into the potential molecular mechanisms that underlie the ability of individual GPCRs (or combinations of GPCRs) to elicit a unique spectrum of responses from their agonist ligands. Further knowledge of these mechanisms will inform drug discovery, since GPCRs are promising therapeutic targets for cancer.

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.

CCN1 and CCN2 are matricellular proteins that are transcriptionally induced by various stimuli, including growth factors. CCN proteins act to facilitate signaling events involving extracellular matrix proteins. Lysophosphatidic acid (LPA) is a lipid that activates G protein-coupled receptors (GPCRs), enhancing proliferation, adhesion, and migration in many types of cancer cells. Our group previously reported that LPA induces production of CCN1 protein in human prostate cancer cell lines within 2-4 h. In these cells, the mitogenic activity of LPA is mediated by LPA Receptor 1 (LPAR1), a GPCR. There are multiple examples of the induction of CCN proteins by LPA, and by the related lipid mediator sphingosine-1-phosphate (S1P), in various cellular models. The signaling pathways responsible for LPA/S1P-induced CCN1/2 typically involve activation of the small GTP-binding protein Rho and the transcription factor YAP. Inducible CCNs can potentially play roles in downstream signal transduction events required for LPA and S1P-induced responses. Specifically, CCNs secreted into the extracellular space can facilitate the activation of additional receptors and signal transduction pathways, contributing to the biphasic delayed responses typically seen in response to growth factors acting via GPCRs. In some model systems, CCN1 and CCN2 play key roles in LPA/S1P-induced cell migration and proliferation. In this way, an extracellular signal (LPA or S1P) can activate GPCR-mediated intracellular signaling to induce the production of extracellular modulators (CCN1 and CCN2) that in turn initiate another round of intracellular signaling.

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.

CCN1/Cyr61 is a secreted extracellular matrix‐associated “matricellular” protein capable of regulating a broad range of cellular activities. Several studies have established that CCN1/Cyr61 is rapidly induced at the transcriptional level when cancer cells are incubated with growth factors that facilitate adhesion signaling, thereby enhancing cancer cell migration and survival. However, the role of CCN1 induction in the proliferative responses to growth factors has not been fully delineated. In this study, we examined the changes in expression levels of CCN1 in response to lysophosphatidic acid (LPA) and epidermal growth factor (EGF) in human prostate and breast cancer cell lines. We also studied the role of free fatty acid receptors (FFARs) in this context. FFAR agonists have been previously reported to show inhibitory effects on growth factor‐mediated proliferation and migration in human prostate and breast cancer cells. PC‐3 and MDA‐MB‐231 cells were serum‐starved for 24 hours and treated with 10uM LPA, 10nM EGF, and +/‐ 1uM TUG‐891. Following the incubation, whole‐cell lysates were collected, and subsequent immunoblotting was performed. LPA and EGF induced CCN1 protein by 4 hours. TUG‐891, an FFA4 agonist, inhibited CCN1 induction by LPA. In parallel, the cells were fixed with 4% paraformaldehyde and CCN1 was visualized using confocal immunofluorescence microscopy. The localization of CCN1 following its induction is consistent with its secretion and subsequent binding to ECM components. These results suggest that CCN1 plays roles in the sequence of downstream responses to growth factors in breast and prostate cancer cells. Support or Funding Information Supported by the ASPET David Lehr Award and by the WSU College of Pharmacy and Pharmaceutical Sciences.