Abstract 4572: Simultaneous activation of C-X-C chemokine receptor 4 and cannabinoid receptor 2 leads to decreased Rho-induced prostate cancer metastasis

BACKGROUND: The binding of the homeostatic chemokine SDF-1 to the G -protein-coupled receptor C-X-C Chemokine Receptor 4 (CXCR4) induces cell migration via heterotrimeric G-proteins of the Gi family. Additionally, activated CXCR4 can stimulate the small GTPase RhoA through Gα13 which is required for directional cell migration, contributing to the metastatic potential of cancer cells. Recently, GPCR hetero-oligomerization has emerged as a means by which new signaling entities can be created, yet how receptor heteromers affect receptor pharmacology remains largely unknown. Here we report that agonist-bound CXCR4 is able to form a heteromer with agonist-bound cannabinoid receptor 2 (CB2) at the cell membrane of prostate cancer (PCa) cells and that receptor interaction inhibits the activation of the cytoskeleton regulator RhoA. EXPERIMENTAL METHODS: To confirm CXCR4-CB2 interactions on the cell membrane, we utilized an in situ proximity ligation assay to detect heterodimer formation on the surface PC-3 cells treated with SDF-1 alone or, simultaneous treatment with SDF-1 and the CB2 agonist AM1241. In order to establish that RhoA activity decreased upon heterodimerization of CXCR4 and CB2, we performed RhoA-GTP pulldown assays in PC-3 cells. We then tested the effects of CXCR4/CB2 heterodimerization on actin cytoskeletal reorganization by performing immunocytochemistry using an anti-Phalloidin antibody to detect F-actin. The ROCK kinase inhibitor Y-27632 was used as positive control of RhoA inhibition in these studies. Next, we assessed the effects of receptor heterodimerization on cellular adhesion to extracellular matrix components as the ability of cancer cells to adhere to the endothelium is critical of transendothelial migration during tumor cell intravasation and extravasation. Intuitively, alterations in adhesion capabilities should affect the ability of PCa cells to migrate through an endothelial cell monolayer. Thus we performed transendothelial migration assays. RESULTS: The proximity ligation assay revealed that CXCR4 and CB2 physically interacted on the cell surface of prostate cancer cells. Furthermore, receptor heterodimerization diminished SDF-1 induced actin cytoskeleton reorganization as characterized by membrane ruffling in a similar manner to the ROCK kinase inhibitor Y-27632. Heterodimer formation, stimulated by simultaneous treatment of cells with SDF-1 and AM1241 also attenuated PCa adhesion and transendothelial migration. CONCLUSION The attenuation of RhoA has multiple functional consequences leading to a less metastatic phenotype. Thus induction of CXCR4-CB2 heterodimerization is a viable pharmacological approach to targeting the CXCR4-RhoA axis as a means of preventing the metastatic dissemination of PCa cells. Citation Format: Kisha A. Scarlett, Christopher J. Coke, Imani Fennell, Cimona V. Hinton. Simultaneous activation of C-X-C chemokine receptor 4 and cannabinoid receptor 2 leads to decreased Rho-induced prostate cancer metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4572.