Abstract 4574: C-X-C chemokine receptor 4 and cannabinoid receptor 2 interact to abrogate CXCL12-mediated cellular response

Expression of C-X-C Chemokine Receptor 4 (CXCR4) encourages tumors cells to migrate to distal organs expressing its cognate ligand, CXCL12, thus facilitating metastasis. Thus, targeting the CXCR4/CXCL12 signaling axis is a good strategy to inhibit the metastatic spread and progression of cancer. It has been suggested that homo- or heterodimerization of GPCRs causes decreased signaling through receptor complexes, representing functional desensitization. In the context of cancer treatment, it is plausible that CXCR4 signaling can be silenced through heterodimeric association with other receptors, thereby inhibiting functions that would lead to metastasis. Various studies suggest that cannabis, signaling via cannabinoid receptors, have anti-proliferative and anti-metastatic properties; though a biochemical mechanism describing how this phenomenon has not been described. We’ve confirmed that agonist-bound CXCR4 and agonist-bound Cannabinoid Receptor 2 (CB2) form a heterodimer that decreases cancer cell migration. Simultaneous treatment of the breast cancer cell line, MDA-MB-231, with CXCL12 and AM1241 (synthetic CB2 agonist), desensitized the intrinsic cellular CXCR4 response of cells to migrate towards CXCL12. Furthermore, through co-immunoprecipitation and proximity ligation assays (PLA), we determined increased interaction between the two receptors upon co-stimulation of respective agonists. To further delineate whether co-stimulation of both receptors led to subsequent heterodimerization, CXCR4 receptor activity was inhibited by an antagonist, AMD3100, or expression of endogenous CXCL12 was downregulated by siRNA. An interaction was observed in simultaneously treated AM1241/CXCL12 cells between CXCR4 and CB2, and surprisingly, in AM1241-treated cells. However, upon pretreatment with AMD3100, and CXCL12-siRNA, receptor association was no longer observed in AM1241-treated cells, supporting the therapeutic notion that treating tumors that endogenously secreted CXCL12 with an exogenous AM1241 can induce dimerization. Moreover, when CXCR4 and CB2 were activated simultaneously with various agonists, decreases in migration were observed, confirming that the regulatory activity was receptor-based, not agonist-based. Further, cells pre-treated with CB2 antagonist (AM630) and combinations of CXCL12 and AM1241 the anticipated decrease in migration was no longer seen. Finally, to determine whether simultaneously-treated, dimerized receptors inhibited activity of respective receptors, calcium mobilization assays showed that transiently activated calcium levels were significantly lower in response to simultaneous treated cells when compared to cells treated with their individual ligands. Therefore, a physical interaction between CXCR4 and CB2 may inhibit the response to CXCL12, leading to a loss in metastatic potential. Citation Format: Christopher Coke, Kisha Scarlett, Kia Jones, Cimona V. Hinton. C-X-C chemokine receptor 4 and cannabinoid receptor 2 interact to abrogate CXCL12-mediated cellular response. [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 4574.