Abstract 4579: Simultaneous activation of C-X-C chemokine receptor 4 and cannabinoid receptor 2 results in decreased cell signaling and tumor cell intravasation

Abstract

Abstract G-protein-coupled receptor (GPCR) heterodimerization has emerged as a means by which new signaling entities can be created with respect to agonist binding. Preliminary data from our lab indicates that agonist-bound GPCR C-X-C Chemokine Receptor 4 (CXCR4) and agonist-bound cannabinoid receptor 2 (CB2R) interact at the cell membrane and that receptor interaction mitigates the effects of CXCL12-mediated CXCR4 signaling. RhoA, Rac1 and Cdc42 are small GTP-binding proteins of the Rho family that are activated downstream of CXCR4 signaling. The effects of RhoA, Rac1 and Cdc42 on the actin cytoskeleton have led to established roles for Rho proteins in cancer cell motility, metastasis and intravasation. Here we report the novel finding that CXCR4/CB2R heterodimerization decreases Rho signaling, resulting in decreased intravasation. To test the effect of receptor heterodimerization on RhoA, Rac1, and Cdc42 activation, PC3 cells were treated with the CXCR4 agonist SDF1α, the CB2R agonist AM1241 or with a combination of SDF1α and AM1241 for 1 minute. Cell lysates were collected and immunoprecipitation experiments were performed to detect RhoA, Rac1 and Cdc42. To test the effects of CXCR4/CB2R heterodimerization on intravsation, we performed a zymography assay for the detection of metalloproteinase-2 (MMP2) and metalloproteinase-9 (MMP9), which are commonly secreted by CXCR4 signaling. Immunopreciptation results indicated an increase in RhoA and Rac1 protein levels after 1 minute in PC3 cells compared to the untreated control. However, when treated with SDF1α and AM1241 for 1 minute, RhoA and Rac1 levels decreased compared to SDF1α independent treatment; there was no observable change in Cdc42 protein levels. The results from the zymography experiments demonstrated that co-treatment with SDF1α and AM1241, which induces CXCR4/CB2R heterodimerization, led to decreased MMP-2 and MMP-9 activity, as compared to the SDF1α-independent treatment. Preliminary results provide novel evidence that CXCR4 and CB2R heterodimerize at the cell membrane, which may result in decreased signaling through RhoA and Rac1, but not through Cdc42. Furthermore, CXCR4/CB2R heterodimerization results in decreased MMP activation, thus abrogating intravsation. Citation Format: Imani N. Fennell, Kisha A. Scarlett, Christopher J. Coke. Simultaneous activation of C-X-C chemokine receptor 4 and cannabinoid receptor 2 results in decreased cell signaling and tumor cell intravasation. [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 4579.