The G-protein-coupled chemokine receptor CXCR4 generates signals that lead to cell migration, cell proliferation, and other survival mechanisms that result in the metastatic spread of primary tumor cells to distal organs. Numerous studies have demonstrated that CXCR4 can form homodimers or can heterodimerize with other G-protein-coupled receptors to form receptor complexes that can amplify or decrease the signaling capacity of each individual receptor. Using biophysical and biochemical approaches, we found that CXCR4 can form an induced heterodimer with cannabinoid receptor 2 (CB2) in human breast and prostate cancer cells. Simultaneous, agonistdependent activation of CXCR4 and CB2 resulted in reduced CXCR4-mediated expression of phosphorylated ERK1/2 and ultimately reduced cancer cell functions such as calcium mobilization and cellular chemotaxis. Given that treatment with cannabinoids has been shown to reduce invasiveness of cancer cells as well as CXCR4-mediated migration of immune cells, it is plausible that CXCR4 signaling can be silenced through a physical heterodimeric association with CB2, thereby inhibiting subsequent functions of CXCR4. Taken together, the data illustrate a mechanism by which the cannabinoid system can negatively modulate CXCR4 receptor function and perhaps tumor progression. G-protein-coupled receptors (GPCRs)2 constitute the largest family of transmembrane receptors (1, 2), and their activation by an appropriate agonist triggers signaling through G-protein ␣ (G␣) and/or ␤␥ subunits (3, 4), leading to context-dependent outcomes. GPCRs have been reported to form homodimers, homomultimers, or heterodimers with related or unrelated GPCRs (5). The resultant heterodimers often generate pharmacological outcomes that are distinct from those of GPCR homodimers. Hence, GPCR heterodimers have become attractive targets for new drug development.The G-protein-coupled chemokine receptor CXCR4 is expressed on the surface of endothelial and epithelial cells of many tissues (6, 7), and upon activation by its agonist, stromal cell-derived 1␣ (SDF1␣), CXCR4 generates signals resulting in processes that favor tissue remodeling such as hematopoiesis, angiogenesis, normal tissue maintenance and development, cell migration, and cell proliferation (8 -19). These functions make CXCR4 a key participant in cancer development, progression, and metastasis (20 -24). Clinically, expression of CXCR4 protein in tumors is used to predict tumor aggressiveness, survival probability, and metastasis-associated mortality (17,20,21,(25)(26)(27)(28). Therefore, developing agents that can inhibit the action of CXCR4 in early and advanced stages of cancer may be effective in preventing and managing metastasis (26).Cannabinoid receptors 1 (CB1) and 2 (CB2) (29, 30) are systems comprising receptors, their agonists (exocannabinoids and endocannabinoids), and enzymes for their metabolism (29,30). CB1 is highly expressed in the brain (31), whereas CB2 is expressed in a variety of other tissues (32-36). The most notable cannabinoid ...

G-protein-coupled receptor (GPCR) heterodimerization has emerged as a means by which alternative signaling entities can be created; yet, how receptor heterodimers affect receptor pharmacology remains unknown. Previous observations suggested a biochemical antagonism between GPCRs, CXCR4 and CB2 (CNR2), where agonist-bound CXCR4 and agonist-bound CB2 formed a physiologically nonfunctional heterodimer on the membrane of cancer cells, inhibiting their metastatic potential However, the reduced signaling entities responsible for the observed functional outputs remain elusive. This study now delineates the signaling mechanism whereby heterodimeric association between CXCR4 and CB2, induced by simultaneous agonist treatment, results in decreased CXCR4-mediated cell migration, invasion, and adhesion through inhibition of the Gα13/RhoA signaling axis. Activation of CXCR4 by its cognate ligand, CXCL12, stimulates Gα13 (GNA13), and subsequently, the small GTPase RhoA, which is required for directional cell migration and the metastatic potential of cancer cells. These studies in prostate cancer cells demonstrate decreased protein expression levels of Gα13 and RhoA upon simultaneous CXCR4/CB2 agonist stimulation. Furthermore, the agonist-induced heterodimer abrogated RhoA-mediated cytoskeletal rearrangement resulting in the attenuation of cell migration and invasion of an endothelial cell barrier. Finally, a reduction was observed in the expression of integrin α5 (ITGA5) upon heterodimerization, supported by decreased cell adhesion to extracellular matrices Taken together, the data identify a novel pharmacologic mechanism for the modulation of tumor cell migration and invasion in the context of metastatic disease. This study investigates a signaling mechanism by which GPCR heterodimerization inhibits cancer cell migration. .

Background: Transcription regulation is essential for angiogenesis, but the role of Irx3 in this process remains to be defined.Results: Irx3 promotes endothelial cell migration and tip cell specification through VEGF-Notch signaling.Conclusion: Irx3 is a novel proangiogenic mediator of endothelial cell migration and cell fate.Significance: Manipulation of Irx3 may provide novel therapeutic strategies in adult vascular pathologies.

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.