Abstract 2512: Agonist-induced heterodimerization between CXCR4 and CB2 inhibits Gα13/RhoA-mediated cell migration

Abstract

Heterodimerization of G protein-coupled receptor exemplifies that GPCRS can change intracellular signaling that would result from each individual receptor. Yet, how heterodimers affect receptor pharmacology remains unknown. We've demonstrated that agonist-bound C-X-C Chemokine Receptor 4 (CXCR4) and Cannabinoid Receptor 2 (CB2) formed a non-functional heterodimer on the membrane of cancer cells, inhibiting their migratory potential in vitro. The impact on the signaling entities responsible for reduced migration upon heterodimerization remain elusive, and therefore, we investigated whether the physical association of CXCR4 and CB2 resulted in decreased signaling from the CXCR4-mediated Gα13/RhoA signaling axis, which leads to phenotypic changes involved in migration. To depict receptor specificity in the role of heterodimer formation on downstream signaling events, we used human siRNA against CXCR4 prior to assaying for Gα13/RhoA immune-complexes to demonstrate that heterodimerization of CXCR4/CB2 decreased RhoA activation with the same potency as genomic knockdown of CXCR4 (PC3). To demonstrate that agonist-induced CXCR4/CB2 heterodimerization attenuated RhoA-dependent cell migration, we immunoblotted for PRG protein expression to determine the heterodimer reduced expression of PDZ-RhoGEF (PRG) which is required for RhoA-dependent cell migration and immunoblotted for LKB1, an establish regulator of RhoA-dependent cell polarity. Finally, to characterize the reduction of RhoA-mediated signaling, we performed both wound healing and transwell migration assays in PC3 cells. In PC3 cells transfected with CXCR4-siRNA, we observed a significant reduction in Gα13/RhoA immune-complexes, which was analogous to samples where CXCR4 and CB2 heterodimerized, suggesting that the physical heterodimer is a viable strategy for antagonizing CXCR4. Immunoblotting techniques revealed that the heterodimer decreased PRG protein expression levels in PC3 cells, compared to cells where CXCR4 signaling was active. The reduction in RhoA signaling upon heterodimerization is directly linked to the propensity of cells to migrate as we saw reduced transformation, reduced wound healing, and reduced migration compared to cells where CXCR4 signaling was active. Our results indicate mechanistic insight into our previous observation that a physical heterodimer reduces cell migration via antagonism of the RhoA pathway. Considering clinical and social support for medicinal cannabinoids in cancer treatment, our current and previous studies mechanistically demonstrate cannabinoid applications and efficiency of using agonists instead of antagonists which currently result in severe immune dysfunction due to the inhibition of CXCR4. This heterodimer can be used as a target for metastasis prevention due to CXCR4 in metastasis and cannabinoids in cancer metastasis treatment and pain management. Citation Format: Kisha Scarlett, Elshaddai White, Christopher Coke, Jada Carter, LaToya Bryant, Cimona V. Hinton. Agonist-induced heterodimerization between CXCR4 and CB2 inhibits Gα13/RhoA-mediated cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2512.