agonist-induced cxcr4 and cb2 heterodimerization inhibits ... · cb2 (cnr2), where agonist-bound...

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Signal Transduction Agonist-induced CXCR4 and CB2 Heterodimerization Inhibits Ga13/ RhoA-mediated Migration Kisha A. Scarlett 1 , El-Shaddai Z. White 1,2 , Christopher J. Coke 1,2 , Jada R. Carter 1,2 , Latoya K. Bryant 1 , and Cimona V. Hinton 1,2 Abstract G-proteincoupled 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 sug- gested 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 in vitro. 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 Ga13/RhoA signaling axis. Activation of CXCR4 by its cognate ligand, CXCL12, stimulates Ga13 (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 Ga13 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 a5 (ITGA5) upon heterodimerization, supported by decreased cell adhesion to extracellular matrices in vitro. Taken together, the data identify a novel pharmacologic mechanism for the modulation of tumor cell migration and invasion in the context of metastatic disease. Implications: This study investigates a signaling mechanism by which GPCR heterodimerization inhibits cancer cell migra- tion. Mol Cancer Res; 16(4); 72839. Ó2018 AACR. Introduction G-proteincoupled receptors (GPCR) represent a large fam- ily of cell surface signaling proteins that are involved in mul- tiple physiologic functions and diseases (1, 2). In classical GPCR signaling, the binding of an agonist to a GPCR induces a conformational change of the receptor that catalyzes the exchange of guanosine diphosphate (GDP) from the Ga sub- unit for guanosine triphosphate (GTP), and the dissociation of Ga from Gb subunits (2). Both Ga and Gb subunit com- plexes stimulate several downstream effectors leading to diverse physiologic outcomes (2, 3). While the conventional theory of GPCR signaling has been that GPCRs can exist and function as monomers, homodimers, or as larger oligomeric complexes, a number of GPCRs have been identied to form functionally relevant heterodimers after translation (47). GPCR heterodi- merization has slowly been accepted as a means by which new signaling entities can be created to amplify or desensitize signaling mechanisms that would otherwise result from each individual receptor leading to diverse pharmacologic outcomes (4). Therefore, GPCR heterodimers have emerged as novel targets for therapeutic development (3, 4, 7, 8). CXCR4 is a chemokine receptor, subclass of GPCR, which is dened by its ability to induce directional migration of cells toward a chemotactic gradient (chemotaxis; refs. 911). The role of CXCR4, and its natural ligand CXCL12, has expanded beyond leukocyte recruitment to include critical processes such as tissue remodeling, angiogenesis, hematopoiesis, cell prolif- eration, and most notably, cell migration (10, 12). Its ability to regulate multiple functions over the lifespan of neoplastic and malignant cells makes CXCR4 a critical driver of tumorigenesis, the progression of cancer, and metastasis (9, 10, 12, 13). Clinically, overexpression of CXCR4 correlates with increased tumor growth, invasion, angiogenesis, metastasis, and thera- peutic resistance while CXCR4 antagonism has been shown to sensitize cancer cells to cytotoxic drugs, reduce tumor growth, and metastatic burden (10, 14), thus making CXCR4 an attrac- tive target for early- and late-stage disease management. 1 Center for Cancer Research and Therapeutic Development, Clark Atlanta University, Atlanta, Georgia. 2 Department of Biological Sciences, Clark Atlanta University, Atlanta, Georgia. Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). Corresponding Author: Cimona V. Hinton, Center for Cancer Research and Therapeutic Development, Department of Biological Sciences, Clark Atlanta University, 4017B Thomas Cole Science Research Center, 223 James P. Brawley Drive SW, Atlanta, GA 30314-4385. Phone: 404-880-8134; Fax: 404-880-6756; E-mail: [email protected] doi: 10.1158/1541-7786.MCR-16-0481 Ó2018 American Association for Cancer Research. Molecular Cancer Research Mol Cancer Res; 16(4) April 2018 728 on October 4, 2020. © 2018 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from Published OnlineFirst January 12, 2018; DOI: 10.1158/1541-7786.MCR-16-0481

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Page 1: Agonist-induced CXCR4 and CB2 Heterodimerization Inhibits ... · CB2 (CNR2), where agonist-bound CXCR4 and agonist-bound CB2 formed a physiologically nonfunctional heterodimer on

Signal Transduction

Agonist-induced CXCR4 and CB2Heterodimerization Inhibits Ga13/RhoA-mediated MigrationKisha A. Scarlett1, El-Shaddai Z.White1,2, Christopher J. Coke1,2,Jada R. Carter1,2, Latoya K. Bryant1, and Cimona V. Hinton1,2

Abstract

G-protein–coupled receptor (GPCR) heterodimerization hasemerged as a means by which alternative signaling entities canbe created; yet, how receptor heterodimers affect receptorpharmacology remains unknown. Previous observations sug-gested a biochemical antagonism between GPCRs, CXCR4 andCB2 (CNR2), where agonist-bound CXCR4 and agonist-boundCB2 formed a physiologically nonfunctional heterodimeron the membrane of cancer cells, inhibiting their metastaticpotential in vitro. However, the reduced signaling entitiesresponsible for the observed functional outputs remain elusive.This study now delineates the signaling mechanism wherebyheterodimeric association between CXCR4 and CB2, inducedby simultaneous agonist treatment, results in decreasedCXCR4-mediated cell migration, invasion, and adhesionthrough inhibition of the Ga13/RhoA signaling axis. Activationof CXCR4 by its cognate ligand, CXCL12, stimulates Ga13(GNA13), and subsequently, the small GTPase RhoA, which

is required for directional cell migration and the metastaticpotential of cancer cells. These studies in prostate cancer cellsdemonstrate decreased protein expression levels of Ga13 andRhoA upon simultaneous CXCR4/CB2 agonist stimulation.Furthermore, the agonist-induced heterodimer abrogatedRhoA-mediated cytoskeletal rearrangement resulting in theattenuation of cell migration and invasion of an endothelialcell barrier. Finally, a reduction was observed in the expressionof integrin a5 (ITGA5) upon heterodimerization, supported bydecreased cell adhesion to extracellular matrices in vitro. Takentogether, the data identify a novel pharmacologic mechanismfor the modulation of tumor cell migration and invasion in thecontext of metastatic disease.

Implications: This study investigates a signaling mechanismby which GPCR heterodimerization inhibits cancer cell migra-tion. Mol Cancer Res; 16(4); 728–39. �2018 AACR.

IntroductionG-protein–coupled receptors (GPCR) represent a large fam-

ily of cell surface signaling proteins that are involved in mul-tiple physiologic functions and diseases (1, 2). In classicalGPCR signaling, the binding of an agonist to a GPCR inducesa conformational change of the receptor that catalyzes theexchange of guanosine diphosphate (GDP) from the Ga sub-unit for guanosine triphosphate (GTP), and the dissociation ofGa from Gb� subunits (2). Both Ga and Gb� subunit com-plexes stimulate several downstream effectors leading to diversephysiologic outcomes (2, 3). While the conventional theory of

GPCR signaling has been that GPCRs can exist and function asmonomers, homodimers, or as larger oligomeric complexes, anumber of GPCRs have been identified to form functionallyrelevant heterodimers after translation (4–7). GPCR heterodi-merization has slowly been accepted as a means by which newsignaling entities can be created to amplify or desensitizesignaling mechanisms that would otherwise result from eachindividual receptor leading to diverse pharmacologic outcomes(4). Therefore, GPCR heterodimers have emerged as noveltargets for therapeutic development (3, 4, 7, 8).

CXCR4 is a chemokine receptor, subclass of GPCR, which isdefined by its ability to induce directional migration of cellstoward a chemotactic gradient (chemotaxis; refs. 9–11). Therole of CXCR4, and its natural ligand CXCL12, has expandedbeyond leukocyte recruitment to include critical processes suchas tissue remodeling, angiogenesis, hematopoiesis, cell prolif-eration, and most notably, cell migration (10, 12). Its ability toregulate multiple functions over the lifespan of neoplastic andmalignant cells makes CXCR4 a critical driver of tumorigenesis,the progression of cancer, and metastasis (9, 10, 12, 13).Clinically, overexpression of CXCR4 correlates with increasedtumor growth, invasion, angiogenesis, metastasis, and thera-peutic resistance while CXCR4 antagonism has been shown tosensitize cancer cells to cytotoxic drugs, reduce tumor growth,and metastatic burden (10, 14), thus making CXCR4 an attrac-tive target for early- and late-stage disease management.

1Center for Cancer Research and Therapeutic Development, Clark AtlantaUniversity, Atlanta, Georgia. 2Department of Biological Sciences, Clark AtlantaUniversity, Atlanta, Georgia.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Cimona V. Hinton, Center for Cancer Research andTherapeutic Development, Department of Biological Sciences, Clark AtlantaUniversity, 4017B Thomas Cole Science Research Center, 223 James P. BrawleyDrive SW, Atlanta, GA 30314-4385. Phone: 404-880-8134; Fax: 404-880-6756;E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-16-0481

�2018 American Association for Cancer Research.

MolecularCancerResearch

Mol Cancer Res; 16(4) April 2018728

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The cannabinoids system consists of lipophilic cannabi-noids (endocannabinoids and exocannabinoids), cannabi-noid receptors (CB1 or CB2) and their enzymes for synthesisand degradation (15–18). Delta-9-tetrahydrocannabinol(D9-THC), the major psychoactive and immunomodulatorycomponent of marijuana, is the most notable cannabinoid(18, 19), and clinically, is used for its antipalliative effects.However, emergent data demonstrates that cannabinoidspossess antiproliferative capabilities and regulate key cellsignaling pathways that promote cell survival, invasion,angiogenesis, and metastasis in various cancer models, pro-pelling cannabinoids to the forefront of cancer therapeuticsresearch (3, 20).

Allosteric interactions within chemokine receptor heterodi-mers inhibit receptor activation in some instances, while otherreceptor pairings cause enhanced signaling and trigger entirelynew responses downstream of receptor activation (3, 21, 22). Forexample, D�ecaillot and colleagues demonstrated that CXC che-mokine receptors CXCR4 and CXCR7 form heterodimers thatenhance cell migration in response to CXCL12 through recruit-ment of b-arrestin to the heterodimeric complex. In addition,extensive evidence points to heterodimerization between chemo-kine receptors with nonchemokine GPCRs or non-GPCR recep-tors (21, 22). In cancer, we (3) and our colleagues (5, 6) dem-onstrated that CXCR4-mediated functions were abrogated bysimultaneous, agonist-induced heterodimerization of CXCR4with GPCRs, such as we have demonstrated with CB2 (3), where-by heterodimers reduced cellular migration and the metastaticpotential of tumor cells. However, the underlying signalingmechanisms governing the observed reduction in cellular migra-tion were not elucidated.

The Rho family of GTPases are small GTP-binding proteinsthat function as molecular switches controlling a wide spec-trum of signal transduction pathways in eukaryotic cells(23–26). They regulate actin cytoskeleton dynamics, and mod-ulate cell polarity, microtubule dynamics, membrane transportpathways, and transcriptional activity (8, 21, 23–25). Expres-sion of the GTPase, RhoA, is implicated in increased tumoraggressiveness in multiple cancer types, and loss of RhoAprevents proliferation and cellular migration both in vitro andmetastasis in vivo (23). Furthermore, the CXCL12/CXCR4 sig-naling axis stimulates RhoA activation through Ga13, leadingto phosphorylation of myosin light chain (MLC) by Rho-associated protein kinase (ROCK), which is required for direc-tional cell migration, the promotion of invasive protrusions,and remodeling of the extracellular matrix (26–28).

We tested whether a physical heterodimeric associationbetween CXCR4 and CB2 results in decreased CXCR4-medi-ated cell migration via reduction of the Ga13–RhoA signalingaxis. First, we analyzed RhoA activation upon heterodimeriza-tion, and expression of downstream targets of RhoA signaling.Second, we investigated alterations to cellular invasion, adhe-sion, and morphology as a result of reduced RhoA expressionupon CXCR4/CB2 heterodimerization. Finally, to correlate therelationship between the effects of CXCR4 heterodimerization,reduced RhoA expression, and the migratory ability of cancercells, we probed for expression of metastatic markers. Weobserved that CXCR4/CB2 heterodimerization antagonizedexpression of Ga13, followed by reduced activity of RhoA andfurther downstream targets. The resultant signaling blockadeinterfered with cancer cells developing a metastatic phenotypic

and functionally migrating through an endothelial cell layer ina RhoA-dependent manner. Altogether, our results furthersupport agonist-induced CXCR4/CB2 heterodimerizationas an emerging approach to inhibiting the metastatic spreadof cancer.

Materials and MethodsCell lines and reagents

Human metastatic prostate cancer cell line (PC3), humanmetastatic breast cancer cell line (MDA-MB-231), and humanembryonic kidney cells (HEK 293T) were purchased from ATCC.PC3 and MDA-MB-231 cells were grown in RPMI1640 andHEK239T cells were grown in DMEM-F-12; all lines were supple-mented with 10% FBS, 1% nonessential amino acids, and 1%antibiotic/antimycotic in a humidified incubator (5% CO2) at37�C. Human umbilical vein endothelial cells (HUVEC) werepurchased from ATCC and maintained in endothelial growthmedium-2 [Lonza; 2% FBS, human endothelial growth factor(hEGF), hydrocortisone, GA-1000 (gentamicin, amphotericin-B),human fibroblast growth factor-B (hFGF-B), R3-IGF-1, ascorbicacid, and VEGF)] in a humidified incubator (5% CO2) at 37�C.Cell lines were authenticated using a short tandem repeat (STR)DNA profiling by ATTC at time of purchase, and every 6 monthsthereafter. Mycoplasma was monitored using the MycoAlertDetection Kit (Lonza). Human CXCR4 agonist, CXCL12(100ng/mL working concentration), was purchased from Pepro-Tech, Inc. Human AM1241 ligand (CB2 agonist; 1 mmol/L) waspurchased from Cayman Chemicals. The CXCR4 antagonist,AMD3100 (1 mg/mL), and Rho-associated kinase inhibitor(ROCK), Y-27631 (10 mmol/L) were purchased from Sigma-Aldrich. Human CXCR4-siRNA and control-siRNA (60 nmol/L)was from Santa Cruz Biotechnology. Prior to treatment withagonists and antagonists, cells were serum-starved for 24 hoursin media only (0% FBS, 0% nonessential amino acids, and 0%antibiotic/antimycotic) for 24 hours in 5%CO2 at 37�C. Samplesdenoted as "control" or "untreated" received fresh media supple-mented with corresponding vehicles (e.g., 0.1%PBS/BSA, DMSO,etc.) for each agonist or antagonist.

RhoA activity assayActivated RhoA was detected using a RhoA Activity Assay Kit

(Cell Biolabs, Inc.) as per the manufacturer's instructions. Briefly,1.5 � 106 cells were cultured to 80% confluence then serum-starved for 24 hours. Cells were pretreated with antagonists for1 hour prior to stimulation with various agonists for 1 minute5%CO2 at 37�C. Cells were harvested as described by the kit, and1 mg of protein from each sample was immunoprecipitatedwith Rhotekin-RBD beads at 4�C overnight with gentle agitation.The beads were pelleted, washed thrice, and boiled in Laemmelisample buffer for 5 minutes. Samples were separated by 15%SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF)membrane, and blocked in 3% BSA in 1� TBST. Rho-A wasdetected by anti-RhoA antibody (1:100) in 3% BSA/TBST over-night at 4�Candharvested forWestern blot analysis. RhoA activitywas normalized to total RhoA protein expression via Westernblot analysis.

siRNA transfectionTransient transfection of CXCR4-specific human siRNA (Santa

Cruz Biotechnology; sc-35421) was performed on PC3 cells using

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Lipofectamine 2000 (Invitrogen). Cells (1 � 106) were plated in10% FBS/RPMI in 100-mm culture dishes and then transfectedwith 60 mmol/L CXCR4-siRNA or scramble/control-siRNA (SantaCruz Biotechnology) inOpti-MEM at 37�C/5%CO2 for 12 hours.Cells recovered in 10% FBS/RPMI for 24 hours, starved for24 hours, followed by treatment with 100 ng/mL CXCL12 and/or 1 mmol/L AM1241, and then were harvested for immunopre-cipitation and/or Western blot analysis.

ImmunoprecipitationA total of 1.5 � 106 cells per sample were serum starved for

24 hours prior to pretreatment with 1 mg/mL AMD3100 or10 mmol/L Y-27632, followed by treatment with 100 ng/mLCXCL12, 1 mmol/L AM1241, or CXCL12 and AM1241 simulta-neously for 1 (PC3 andHEK 293) or 10 (MDA-MB-231)minutes.Cells were lysed in Rho A lysis buffer from above, sonicated, thenfollowed by incubation on ice for 30 minutes. Lysates werecentrifuged at maximum speed for 10 minutes at 4�C, then500–750 mg of protein from each sample was incubated withGa13 antibody (1:100; Abcam) overnight at 4�C on a rockingplatform. Protein A/G PLUS Agarose Beads (1:10, Santa CruzBiotechnology) were added to each sample for 2 hours at 4�Cwith gentle agitation. Cell lysates were washed twice in 1� PBS(maximum speed for 5 minutes at 4�C), boiled inLaemelli sample buffer for 5 minutes, followed by 12%SDS-PAGE. Proteinwas transferred to aPVDFmembrane, blockedin 5% milk/TBST, immunoblotted in 5% BSA/TBST with ananti-RhoA (1:1,000; Cell Signaling Technology). Primaryantibodies were detected with corresponding horseradishperoxidase (HRP)-conjugated secondary antibodies (1:1,000;Jackson) and enhanced chemiluminescence (Luminata; ThermoFisher Scientific).

Antibodies and Western blottingPDZ-RhoGEF and LKB1 antibodies were purchased from

Santa Cruz Biotechnology. F-actin was purchased from Abcam.Phosphorylated myosin light chain (pMLC), integrin aV, andintegrin b3 were purchased from Cell Signaling Technology. Atotal of 2.0 � 106 cells per sample were serum starved for24 hours prior to pretreatment with 1 mg/mL AMD3100 or10 mmol/L Y-27632 followed by treatment with 100 ng/mLCXCL12, 1 mmol/L AM1241, or CXCL12 and AM1241 simul-taneously for various time points. Cells were lysed and soni-cated in 1� Cell Signaling Technology lysis buffer prior toincubation on ice for 30 minutes. Lysates were centrifuged atmaximum speed for 10 minutes at 4�C, and then equalamounts of protein per sample were separated by SDS-PAGEand transferred to PVDF membrane. Protein-bound mem-branes were blocked in 3% BSA/1� TBST, (1� TBST for pMLC),and subsequently incubated with primary antibodies: RhoA(1:500, Cell Signaling Technology); F-actin (1:1,000, Abcam);pMLC (1:500, Cell Signaling Technology); LKB1 (1:100, SantaCruz Biotechnology); integrin a5 (1:1,000, Cell SignalingTechnology); integrin b3 (1:1,000, Cell Signaling Technology);total AKT (1:1,000, Cell Signaling Technology); or total ERK1/2(1:1,000, Cell Signaling Technology) overnight at 4�C in 3%BSA/TBST; (1� TBST alone for pMLC). Primary antibodies weredetected by HRP-conjugated secondary antibodies at 1:5000–1:10,000 diluted in 3% BSA/1� TBST (1� TBST alone forpMLC) and enhanced chemiluminescence (SuperSignal WestPico Chemiluminescent Substrate or Lumniglo).

Filamentous (F)-actin immunocytochemistryCells were seeded on glass coverslips at a density of 2 � 105

cells/well and incubated for 72 hours at 5% CO2 at 37�Cuntil they were approximately 80% confluent. Cells were thenserum starved for 24 hours, then pretreated with 1 mg/mL ofAMD3100 or 10 mmol/L of Y-27632 for 1 hour prior totreatment with 100 ng/mL CXCL12 or 1 mmol/L AM1241 eachalone, or combined CXCL12/AM1241 at 5% CO2 and 37�Cin serum-free media for 90 minutes. Coverslips were rinsedtwice with ice-cold 1� PBS, fixed with 4% paraformaldehydefor 10 minutes, and washed again with 1� PBS. Cells wereblocked with 1% BSA/TBST and incubated with FITC-conju-gated phalloidin (100 nmol/L in 1� PBS, Enzo Life Science)to detect F-actin for 1 hour at room temperature with gentleagitation. Cells were then washed thrice with 1� PBS andcounterstained with DAPI for nuclear visualization. Slides werevisualized and images were captured using a Zeiss LSM 700confocal microscope. Scale bar ¼ 50 mm.

Cell migrationMDA-MB-231 cells (1 � 106 per sample) were plated in p-100

tissue culture plates and serum-starved for 24 hours. Cells weredetached with 1� Citric Saline, washed in 1� PBS, pelleted bycentrifugation at 3,000 � g for 5 minutes, and then counted toprepare 50,000 cells per sample in 150 mL of media. Transwellinserts were prepared by precoating the outer membrane with50 mL rat tail collagen (50 mg/mL in 0.02N acetic acid; BDBiosciences) for cell adherence to the transwell insert, and incu-bating at 4�C overnight. The next day, the inner membrane wascoated as above, and incubated for 1 hour at room temperature.Inserts were washed thrice in 1� PBS prior to use for assay.Treatment (100 ng/mL CXCL12 and 1 mmol/L AM1241 each orin combination, 1 mg/mL AMD3100 or 10 mmol/L Y-27632) werecalculated for a final concentration of 500 mL each, but preparedin 350 mL per sample, and placed into the bottom well of eachchamber. Fifty-thousand cells per sample were resuspended in150 mL of media and were seeded into the transwell insert of24-well transmigration chambers (8-mmpore; BD Falcon), bring-ing the total volume of each chamber to 500 mL. To allow formigration, plateswere incubated in 5%CO2and37�C for 6hours,and the inner-top chambers were cleaned with a cotton swab.Cells attached to the outer-top chambers were fixed with 10%formalin for 15 minutes at room temperature, washed thrice in1 � PBS, stained with 0.05% crystal violet for 15 minutes, thenwashed 6 times in water. Five representative fields of each insertwere counted under a light microscope, and the migration indexwas calculated and graphed as the x-fold change in migrationobserved over control cells or CXCL12-treated cells. Experimentswere performed at least thrice, and significant significance wasanalyzed via GraphPad Prism (�, P < 0.05; ��, P < 0.001).

Transendothelial migration assayMigration though an endothelial cell barrier was performed

via a Transwell Migration Assay Kit (Cell Biolabs, Inc.) per themanufacturer's instructions. Briefly, 1 � 105 HUVECS per wellwere seeded inside of Transwell inserts in 500-mL completeEGM-2 media at 5% CO2 and 37�C for 72 hours to allowformation of a cell monolayer. In the interim, 1 � 106 of PC3,MDA-MB-231, or HEK293T cells were preincubated with1� CytoTracker (kit provided) allowing each cell to becomefluorescently labeled, then were pelleted and washed 3� in

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serum-free media and resuspended in 500 mL of serum-freemedia. EGM-2 media was removed from inside each HUVECtranswell and replaced with 300 mL each of PC3, MDA-MB-231, or HEK293T serum-free RPMI cell suspension. Transwellinserts were transferred to fresh plates containing serum-freemedia supplemented with combinations of 100 ng/mLCXCL12, 1 mmol/L of AM1241, 1 mg/mL of AMD3100, and/or10 mmol/L Y-27632 and the plate was incubated in 5% CO2 at37�C for 24 hours. Fluorescently labeled cells that transmi-grated were lysed in 1� Cell Lysis Buffer (Cell Biolabs, Inc.) andanalyzed using a plate reader at OD 480 nm/520 nm. Exper-iment was repeated thrice, and statistical significance wasanalyzed via GraphPad Prism (�, P < 0.05; ��, P < 0.001).

Wound healing assayA total of 1 � 106 cells per sample were seeded in a 6-well

culture plate overnight, and then serum-starved for 24 hours.Cells were pretreated with 1 mg/mL of AMD3100 or 10 mmol/Lof Y-27632 for 1 hour, removed, and then plates were stored onice. One milliliter of 1� PBS was added to each well, a verticalwound was made across each well using a micropipette tip, andnonadherent cells were removed by washing with 1� PBS.Serum-free media with supplements of 100 ng/mL CXCL12,and 1mmol/L of AM1241 each or in combination, along with1 mg/mL AMD3100, or 10 mmol/L Y-27632. Wound closure wasallowed for up to 6 hours at 5% CO2 and 37�C, with picturescaptured at 0, 2, 4, and 6 hours. The wound area of each samplewas quantified using ImageJ software. Experiments were repeat-ed thrice; statistical significance was analyzed via GraphPadSoftware (�, P < 0.05; ��, P < 0.001).

Extracellular matrix adhesion assayThe adhesion of cells to components of the extracellular

matrix (ECM) was quantified using the CytoSelect AdhesionAssay (ECM Array, Colorimetric Format; Cell Biolabs, Inc.). Atotal of 1.5 � 106 cells per sample were seeded and grown toapproximately 80% confluence. Cells were serum-starved for 24hours and then harvested by 0.25% trypsin/EDTA for 5 min-utes. Trypsin was removed after centrifugation (2,000 � g for 5minutes) and cells were resuspended in serum-free RPMI at aconcentration of 1.0 � 106 cells/mL. The cell suspensionwas preincubated with 1 mg/mL AMD3100 or 10 mmol/LY-27632 in serum-free media for 1 hour in 5% CO2 at 37�C.Thereafter, additional treatments of 100 ng/mL of CXCL12alone, 1 mmol/L of AM1241 each, or combination was addedto the cell suspension. A total of 150 mL of each cell suspensionwas plated onto a 48-well culture plate precoated with variousECM components (fibronectin, collagen I, and laminin I), andthen incubated for 2 hours in 5% CO2 at 37�C to allowattachment. Cells were washed carefully with 1� PBS to removeunbound cells, and adherent cells were incubated in 200 mL ofCell Stain Solution for 10 minutes on an orbital shaker (CellBiolabs, Inc). Thereafter, each well was gently washed 4� with500 mL of double deionized water, followed by incubation in200 mL Extraction Solution (Cell Biolabs, Inc.) for 10 minuteson an orbital shaker. Following extraction, 150 mL of eachsample was transferred to a 96-well plate and measured usinga spectrophotometer at OD 560 nm. Each experiment wasrepeated thrice and statistical significance via GraphPad Prism(�, P < 0.05; ��, P < 0.001).

Statistical analysisStudent t test, two-way ANOVA, or Bonferroni multiple

comparison test were performed to determine statistically signi-ficant differences between groups. Statistical analysis was per-formed via GraphPad Prism.

ResultsSimultaneous treatment with CXCL12 and AM1241 inhibitsGa13/PRG–mediated activation of RhoA

We have shown formation of a physical heterodimerbetween CXCR4 and CB2 upon simultaneous (combined)treatment with agonists CXCL12 and AM1241, respectively(3). This heterodimer between CXCR4 and CB2 impededcellular migration; however, a mechanism was not defined.Independent studies have demonstrated that CXCR4-mediatedmigration occurs via the RhoA–PRG–Ga13 pathway (27, 28).Therefore, we first investigated whether heterodimerizationof CXCR4/CB2 decreased RhoA activation in the malignanthuman prostate cancer cells (PC3). Isolating active forms(GTP-bound) of RhoA with Rhotekin-glutathione beadsdemonstrated that CXCL12 caused an increase in expressionof RhoA-GTP compared with control (untreated) cells (Fig. 1A).However, simultaneous treatment with CXCL12 and AM1241,which induced the heterodimer (3), resulted in decreasedprotein expression of RhoA-GTP, indicating a decrease in acti-vation of RhoA compared with CXCL12-stimulated cells(Fig. 1A). The decrease in activated Rho-A expression levelswas consistent in cells pretreated with AMD3100 (CXCR4antagonist) or Y-27632 (RhoA/ROCK inhibitor) prior to treat-ment with CXCL12 (Fig. 1A).

Tan and colleagues demonstrated that CXCR4-mediated acti-vation of RhoA, and resulting directional cellular migration, wasspecifically mediated by the small G protein Ga13 (27). Consis-tent with the pattern for RhoA activity, we observed that CXCL12led to increased expression Ga13, as determined by expression ofRhoA via Ga13/RhoA immune complexes, compared withuntreated cells in PC3 and the human breast cancer cell line,MDA-MB-231 (Fig. 1B and C). Conversely, simultaneous stimu-lation of PC3 or MDA-MB-231 cells with CXCL12 and AM1241resulted in decreased expression of Ga13, which was consistentwith expression levels of Ga13 in cells treated with AMD3100 orY-27632 (Fig. 1B and C).

To support our premise that a dimer between CXCR4 andCB2 inhibits the action of CXCR4 to activate Ga13-RhoA, weused the human embryonic kidney cell line, HEK 293T, as we(3) and others have published is CXCR4-null. Pretreatmentwith the RhoA/ROCK inhibitor Y-27632 visibly reducedGa13/RhoA immune complexes. However, RhoA protein levelsdisplayed consistent expression among all other treatmentgroups, with no visible induction by CXCL12 (Fig. 1D); thebasal level of Ga13–RhoA immune complexes could have beenmediated by any other GPCR. Likewise, we used siRNA target-ing CXCR4 to further show that the dimer between CXCR4 andCB2 has similar antagonistic effects as would a knockdown ofthe CXCR4 receptor (Fig. 1E). In PC3 cells, we observedreduction of Ga13/RhoA immune complexes in CXCL12-trea-ted cells transfected with CXCR4-siRNA compared with cellswithout siRNA. However, the reduction in Ga13/Rho Aimmune complexes in cells was more robust in cells treatedCXCL12 and AM1241 (both with and without siRNA),

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suggesting that the heterodimer is an efficient way to manageCXCR4 signaling (Fig. 1E).

In breast cancer cells stimulated with CXCL12, Struckhoffand colleagues demonstrated that PDZ-RhoGEF (PRG)guanine nucleotide exchange factor was required for RhoA-dependent cell migration (28). We, too, observed that CXCL12increased PRG protein expression levels in PC3 cells (Fig. 1C).Contrastingly, when PC3 cells were treated with CXCL12and AM1241 simultaneously, expression levels of PRG pro-tein expression decreased compared with that of CXCL12treatments, further supporting that agonist-induced heterodi-merization is an effective mechanism to antagonize CXCR4-mediated signaling that leads to cell migration. As a control,PC3 cells treated with AMD3100 or Y-27632 virtually abol-ished PRG protein expression in the presence of CXCL12(Fig. 1F), indicating that the observed results were specificallymediated by CXCR4–RhoA signaling. Collectively, coincidingwith our previously observed decrease in cellular migration(3), an induced GPCR heterodimer between CXCR4 and CB2antagonizes CXCR4-mediated cell migration via the RhoA–Ga13–PRG pathway.

Agonist-induced CXCR4/CB2 heterodimerization inhibitsRhoA-mediated cytoskeleton reorganization and attenuatesRhoA-dependent cell migration

RhoA plays a critical role in cell migration through itsintimate relationship with the actin cytoskeleton, which haslong been implicated in generating the contractile forcesneeded for membrane blebbing, lamellae formation, andmembrane ruffling at the leading edge and retraction of thetrailing edge of migratory cells (29–32). To evaluate whetherour induced heterodimer could inhibit RhoA-mediatedchanges in the actin cytoskeleton that are necessary for migra-tion, we first examined phenotypic changes in filamentousactin (F-actin) in response to independent (CXCL12) andcombinatory (CXCL12/AM1241) agonist stimulation in PC3and MDA-MB-231 cell lines. CXCL12 produced expected pro-migratory alterations in cell morphology such as membraneruffling, lamellae formation, and membrane protrusions incancer cell lines compared with untreated cells that remainedcharacteristically smooth at the cell membrane (Fig. 2 and 3).However, cells simultaneously stimulated with CXCL12and AM1241 also exhibited a smooth cell membrane, similar

- + - + + +- - + + - -- - - - + -- - - - - +

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Figure 1.

CXCR4/CB2 heterodimerization attenuates Ga13/PRG–mediated RhoA activation in PC3 cells. A, Serum-starved PC3 cells were pretreated for 1 hourwith Y-27632 or AMD3100 followed by treatment with CXCL12 for 1 minute. Alternatively, PC3 cells were treated for 1 minute with CXCL12 or AM1241alone, or CXCL12/AM1241 simultaneously. Lysates were incubated with Rhotekin-RBD beads prior to harvesting for immunoblotting for RhoA-GTPprotein expression; total RhoA represents lysate prior to pull-down with Rhotekin-RBD beads. PC3 cells (B), MDA-MB-231 cells (C), and HEK 293T cells (D)were treated as described in Materials and Methods, and then the lysates were incubated with anti-Ga13 antibody overnight at 4�C followed bystandard immunoprecipitation techniques. Isolated protein lysates were immunoblotted for RhoA protein expression; input represents lysate prior toimmunoprecipitation. E, PC3 were transfected with 60 mmol/L human siRNA targeting CXCR4 or nonspecific control siRNA for 12 hours, followed byrecovery in 10% FBS/RPMI for 24 hours. Cells were then harvested for Ga13/RhoA immunoprecipitation or Western blotting. Total ERK1/2 and Total AKTwere used as: (i) loading input controls to demonstrate that siRNA was specific; and (ii) to demonstrate that equal amounts of protein were used forassay. F, Serum-starved PC3 cells were treated as described above, and whole protein lysates were immunoblotted for PRG protein expression;a-tubulin was used as a loading control. Each experiment was performed at least twice. � , P < 0.05; �� , P < 0.001.

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to untreated cells, and indicated little-to-no changes in cyto-skeletal rearrangement; this same phenotype was observed forcells pretreated with AMD3100 (Fig. 2 and 3). Contrastingly,

pretreatment with Y-27632 resulted in apoptotic phenotypeswith an overall decrease in volume of cell cytoplasm andmembrane blebbing in PC3 and MDA-MB-231 cells (Fig. 2

Untreated

Membrane ruffling

CXCL12 AM1241

CXCL12 + AM1241 Y-27632 + CXCL12 AMD3100 + CXCL12

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Figure 2.

Agonist-induced CXCR4/CB2heterodimerization inhibitsRhoA-mediated cytoskeletonreorganization. Serum-starved PC3cells were pretreated for 1 hour withY-27632 or AMD3100 prior totreatmentwith CXCL12 for 90minutes.Subsequently, cells were treated for90 minutes with CXCL12 or AM1241alone, or CXCL12/AM1241simultaneously. Samples were fixedand stained with FITC-phalloidin tovisualize F-actin; nuclei werecounterstained with DAPI. Arrowshighlight areas of lamellae formationand membrane ruffling.

Membrane ruffling

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AM1241 CXCL12 + AM1241 Y-27632 + CXCL12 AMD3100 + CXCL12

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Figure 3.

Agonist-induced CXCR4/CB2 heterodimerization inhibits RhoA-mediated cytoskeleton reorganization. Serum-starved MDA-MB-231 cells were pretreatedfor 1 hour with Y-27632 or AMD3100 prior to treatment with CXCL12 for 90 minutes. Subsequently, cells were treated for 90 minutes with CXCL12 orAM1241 alone, or CXCL12/AM1241 simultaneously. Samples were fixed and stained with FITC-phalloidin to visualize F-actin; nuclei were counterstainedwith DAPI. Arrows highlight areas of lamellae formation and membrane ruffling.

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and 3), which is confirmed in literature (33, 34). In HEK 293Tcells, as expected, we did not observe phenotypic changesin response to CXCL12, AM1241, or the agonist combined(Fig. 4A) as they are CXCR4-null.

We examined protein expression levels of F-actin to support thephenotypic changes observed above where expression of F-actinin PC3 cells, simultaneously stimulated with CXCL12 andAM1241,was comparablewith untreated cells, further supportingthat the CXCR4/CB2 heterodimer prevents the promigratoryphenotype necessary for cellular migration (Fig. 4B). Likewise,AMD3100 and Y-27632 displayed markedly reduced F-actinprotein levels (Fig. 4B). RhoA regulates actomyosin contractionin part by stimulating phosphorylation of myosin light chain (p-MLC; refs. 27, 28, 35) which is also required for phenotypicchanges associated with cellular migration. Therefore, we deter-mined whether agonist-induced CXCR4/CB2 heterodimerizationled to downstream attenuation of p-MLC expression in PC3 cells.CXCL12 increased expression of p-MLC protein levels comparedwith the untreated control; however, protein levels of p-MLC insimultaneously treated samples were reduced compared withboth untreated control and CXCL12-treated cells (Fig. 4C). Takentogether, these results further support that an induced CXCR4/CB2 heterodimer effectively attenuates signaling pathways thatleads to a migratory phenotype in cancer.

Failed directional migration is usually a consequence of lossof cell polarity as marked by decreased expression of humantumor suppressor and serine-threonine kinase LKB1 (29).Therefore, we harvested treated samples and immunoblottedfor LKB1, an established regulator of RhoA-dependent cellpolarity (30, 31). Simultaneous stimulation of PC3 cells withCXCL12 and AM1241 decreased protein expression levels ofLKB1 compared with untreated control and CXCL12, as didtreatment with Y-27632 and AMD3100 (Fig. 4D), indicative ofreduced polarity for migration.

To attribute the reduction in RhoA-mediated signaling, as aresult of heterodimerization, to migratory behavior and events(Fig. 5A and B), we observed that CXCL12 significantly stim-ulated wound closure of PC3 cells compared with untreatedcells (Fig. 5B, P ¼ 0.0462). CXCL12 and AM1241 combinedwere deficient in stimulating migration across the wound areawhen compared with CXCL12 alone (Fig. 5A and B; P ¼ 0.042).Likewise, pretreatment of cells with AMD3100 (P ¼ 0.0099) orY-27632 decreased wound closure efficiency compared withCXCL12, lending more credence that GPCR heterodimerizationattenuates RhoA signaling associated with cancer-progressingactivities.

Via simple Transwell migration, CXCL12-treated MDA-MB-231 cells exhibited significantly increased migration compared

Untreated CXCL12 AM1241

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Figure 4.

Agonist-induced CXCR4/CB2 heterodimerization inhibits RhoA-mediated cytoskeleton reorganization. A, Serum-starved HEK 293T cells were pretreatedfor 1 hour with Y-27632 or AMD3100 prior to treatment with CXCL12 for 90 minutes. Subsequently, cells were treated for 90 minutes with CXCL12 orAM1241 alone, or CXCL12/AM1241 simultaneously. Samples were fixed and stained with FITC-phalloidin to visualize F-actin; nuclei were counterstainedwith DAPI. Arrows highlight areas of lamellae formation and membrane ruffling. B–D, Serum-starved PC3 cells were pretreated with Y-27632 orAMD3100 followed by treatment with CXCL12, AM1241, or agonists simultaneously for 90 minutes. Whole protein lysates were analyzed for F-actin (B),pMLC (C), or LKB1 (D). Each experiment was performed at least twice.

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with untreated migrating cells (P ¼ 0.043; Fig. 5C and D).Likewise, CXCL12 and AM1241 combined exhibited signifi-cantly decreased migratory capacity compared with CXCL12alone (P < 0.0001; Fig. 5C and D). In addition, both Y-27632(P < 0.0001) and AMD3100 (P < 0.0001) significantlyreduced transmigration when compared with cells migratingtoward CXCL12 alone (Fig. 5C and D). To effectively metas-tasize from a primary tumor site to a distal organs, aggressivecancer cells must demolish and migrate through an endothelialcell barrier, which is characteristic of intravasation and extrav-asation in vivo (25, 32), and CXCL12-driven transendothelialmigration of metastatic tumor cells has been shown to be Rho-dependent (20, 33, 34). As such, we observed that CXCL12resulted in significantly increased transmigration comparedwith untreated transmigrating cells (P ¼ 0.0296; Fig. 5E).Likewise, CXCL12 and AM1241 combined exhibited signifi-cantly decreased transendothelial migratory capacity com-pared with CXCL12 alone (P ¼ 0.0476; Fig. 5E). In addition,

both Y-27632 (P ¼ 0.0054) and AMD3100 (P ¼ 0.0187)significantly reduced transmigration when compared with cellsmigrating toward CXCL12 alone (Fig. 5E).

Inhibition of CXCR4-mediated signaling viaheterodimerization results in decreased integrin expressionand RhoA-mediated adhesion to the extracellular matrix

Rho GTPase molecules regulate cell adhesion (36, 37) furtherimplicating its role in tumor cell invasion and migration(26). Multiple studies report that CXCR4 signaling leads toenhanced expression of a5 integrin and b3 integrin, whichtrigger persistent adhesion of tumor cells to immobilizedlaminin, collagen, and fibronectin (38). In PC3 cells, CXCL12alone stimulated increased expression levels of a5 integrincompared with the untreated control, while AM1241 in com-bination with CXCL12 reduced expression (Fig. 6A). Althoughwe observed an increase in b3 expression upon CXCL12treatment, which aligns with observations in literature,

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Figure 5.

CXCR4/CB2 heterodimerization attenuates RhoA-dependent cell migration and wound closure. A and B, Serum-starved PC3 cells were pretreated for1 hour with Y-27632 or AMD3100 followed by treatment with CXCL12 and/or AM1241 for up to 6 hours as described in Materials and Methods. ImageJwas used to measure the width of each wound at 0 and 6 hours. Experiments were performed thrice. � , P < 0.05; �� , P < 0.001. C and D, 5 � 104 cellseach were seeded into the top Transwell chamber and allowed to migrate toward combinations of agonists and chemical inhibitors in the bottomchamber for 6 hours at 37�C, 5% CO2. Five fields of each Transwell insert were randomly selected and counted for migrated cells at 10� magnificationusing a Zeiss Axiovert 200M light microscope, and results were analyzed via GraphPad Prism. Experiments were repeated thrice, and data represent theaverage of three independent experiments. D, A graphical representation of total migrated cells. Data represents the average of three independentexperiments; �, P < 0.05; ��, P < 0.001. E, HUVECs were seeded in the top chamber of Transwell inserts allowing for the formation of a confluent monolayer.Fluorescently labeled, serum-starved PC3 cells were added on top of HUVECs in the top chamber, and then, the combinations of agonists and chemicalinhibitors were added to the bottom chambers followed by a 24-hour incubation. Fluorescent invasive cells were quantified at OD 480 nm/520 nm.Experiments were performed thrice (� , P < 0.05; �� , P < 0.001).

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heterodimerization of CXCR4 with CB2 caused no significantlyobservable reduction of b3 protein levels when compared withCXCL12; chemical inhibition with AMD3100 and Y-27632resulted in reduced levels of both a5 integrin and b3 integrin(Fig. 6A).

Finally, we confirmed whether CXCR4/CB2 heterodimeriza-tion regulates adherence to components of the extracellularmatrix (ECM). Using in vitro models of ECM components,we observed that CXCL12 led to a significant increase inadhesion to fibronectin (P ¼ 0.0484), collagen I (P ¼0.0287) and laminin I (P < 0.0001) in PC3 cells when com-pared with untreated cells (Fig. 6B–D). CXCL12 and AM1241combined significantly reduced cellular adhesion to fibronectin(P ¼ 0.0171), collagen I (P ¼ 0.0354), and laminin I (P ¼

0.0317) compared with CXCL12 alone; likewise, Y-27632(fibronectin; P ¼ 0.0154), (collagen I; P ¼ 0.0031), and(laminin I; P ¼ 0.005), and AMD3100 (fibronectin; P ¼0.0322), (collagen I; P ¼ 0.0086), and (laminin I; P ¼0.0264), both yielded reduced adhesion of cells to ECM com-ponents (Fig. 6B–D). These results suggest that a physicalCXCR4/CB2 heterodimer is sufficient to decrease tumor celladhesion, likely due to decreased expression of integrin alphaand subsequent inhibition of RhoA activation.

DiscussionOur results provide mechanistic insight into our previous

observation that a physical CXCR4/CB2 heterodimer reduces

Figure 6.

Inhibition of CXCR4-mediated signaling via heterodimerization results in decreased integrin expression and RhoA-mediated adhesion to the extracellularmatrix. A, Serum-starved PC3 cells were pretreated for 1 hour with Y-27632 or AMD3100 followed by treatment with CXCL12 or AM1241 alone, orCXCL12/AM1241 simultaneously for 6 hours. Cell lysates were harvested for integrin a5 and integrin b3 protein expression; a-tubulin was used as aloading control. Experiment was performed at least twice. B–D, Cells were serum-starved and pretreated Y-27632 and AMD3100 for 1 hour prior to theaddition of CXCL12 or AM1241 alone, or CXCL12/AM1241 simultaneously on matrix-coated plates for 2 hours, stained with Cell Stain Solution (Cell Biolabs, Inc.),and then analyzed at OD 560 nm. Each assay was performed at least thrice. � , P < 0.05; �� , P < 0.001.

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cell migration via antagonism of the RhoA pathway (Fig. 7Aand B). CXCL12 binds to CXCR4 and activates RhoA specif-ically through activation of the small G protein, Ga13 (27),which leads to the subsequent activation of PRG, and isrequired for directional cell migration (ref. 35; Fig. 7A). Onceactivated, RhoA induces changes in the actin cytoskeletonthrough phosphorylation of myosin light chain (26, 27),increased F-actin protein levels, and changes in integrinexpression (39–41). Molecular signaling events initiated bythe CXCL12/CXCR4 cascade enhances cellular functionalityby increasing cell migration and adhesion, ultimately contrib-uting to tumor progression (Fig. 7A). We demonstrated thatthe physical heterodimer between CXCR4/CB2 reduced Ga13recruitment of PRG and subsequent RhoA activation (Fig. 7B).We also demonstrated that reduced migration, adhesion, andother coordinating events that encourage tumor cell migrationwas specifically attributed to the RhoA pathway as reductionupon heterodimerization was parallel to treatment with RhoA/ROCK inhibitor, Y-27632.

RhoGTPases are key regulators of cytoskeletal dynamics thatlead to cell adhesion, polarity, and directional movement.Across several studies, CXCR4 was shown to induce cell migra-tion via signaling through Rho GTPases, which therefore sug-gests this group of proteins as a treatment target for tumors thatexpress CXCR4 (27, 42). During cell movement, Rho is impor-tant for regulating the formation of contractile actin–myosinfilaments and stress fibers, and maintaining focal adhesions atthe rear of the migrating cells, whereas Rac is involved informing actin-rich membrane ruffles, or lamellipodia forma-tion, at the leading edge of the migrating cells, which isrequired to determine directional movement (42). Therefore,the collective events resulting in Rho GTPases represent a keyregulatory event for the intentional chemotaxis of cancer cells(27). Our findings, that heterodimerization reduced CXCR4-mediated RhoA signaling and phenotypic changes associatedwith cell migration, may have broad implications as theCXCL12/CXCR4 signaling axis is critical to a successful meta-static tumor.

Figure 7.

Simultaneous agonist-induced CXCR4/CB2 heterodimerization inhibits RhoA-mediated cell migration and adhesion. A, The binding of CXCL12 to its cognatereceptor, CXCR4, initiates the activation of Ga13 and its dissociation from Gbg subunits. Ga13 directly activates the DBl family RhoGEF, PDZ-RhoGEF(PRG), which subsequently activates the small GTPase, RhoA. The downstream target of RhoA, Rho-associated protein kinase (ROCK), phosphorylatesmyosin light chain (MLC) leading to directional cell migration, the formation of invasive protrusions including membrane ruffling, and increased adhesionto the extracellular matrix. Integrin signaling through an alternate pathway can modulate RhoA activity through activation of focal adhesion kinase 1 (FAK1)that activates RhoA through Ras protein–specific guanine nucleotide-releasing factor 1 (RASGRF1). B, Agonist-induced heterodimerization of CXCR4 and CB2inhibits Ga13 activation and dissociation from Gbg subunits. As a result, PRG ineffectively activates RhoA, which in turn, fails to mediate phosphorylation ofmyosin light chain leading to abrogation of cell migration, invasion, and adhesion. Signaling through alternate pathways, including integrin-mediatedsignaling, is insufficient to overcome inhibition caused by heterodimerization.

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Receptor dimerization is becoming a key paradigm in GPCRbiology and cancer therapeutics. It has been reported for mostaspects of GPCR function: trafficking, internalization, andpharmacologic antagonism and signal transduction (3, 36,43–45). Although the functional consequences of receptorheterodimerization are still emerging, physical heterodimersamplify or antagonize signals that would otherwise result fromeach receptor individually, and change the signaling profileor direction to favor one downstream pathway over another(43, 44). When CXCR4/CB2 heterodimerize, the receptor pairreduces signals that would come from CXCR4 alone, stronglysupporting our global results that inhibiting the action ofCXCR4 whether via: (i) induced receptor heterodimerization;(ii) chemical antagonism of CXCR4 with AMD3100; or (iii)reduced translation of CXCR4 protein via siRNA will reducethe propensity of CXCR4-mediated signaling to activate cellmigration.

GPCR heterodimers also alter ligand selectivity wherephysical heterodimerization resulted in negative bindingcooperativity—only one chemokine ligand binds with highaffinity to the receptor dimer pair (36–38). Considering theoverwhelming clinical and social support for medical canna-binoids in cancer treatment, our current and previous (3)studies mechanistically demonstrate cannabinoid applica-tions, and opens avenues for considering antagonizing CXCR4via induced CXCR4/CB2 heterodimers to slow the metastaticprocess. What's more, we use agonists, instead of antagonists,which currently result in severe immune dysfunction due toinhibition of CXCR4 (46). The established role of CXCR4 inmetastasis and the broad role for cannabinoids in cancermetastasis treatment, pain management, palliative care,bioavailability, and noninvasive administration (47, 48)

present this specific heterodimer as a target for metastasisintervention.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: K.A. Scarlett, C.V. HintonDevelopment of methodology: K.A. Scarlett, E.Z. White, C.V. HintonAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K.A. Scarlett, E.Z. White, J.R. Carter, L.K. Bryant,C.V. Hinton, C.J. CokeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K.A. Scarlett, E.Z. White, J.R. Carter, L.K. Bryant,C.V. Hinton, C.J. CokeWriting, review, and/or revision of the manuscript: K.A. Scarlett, E.Z. White,C.V. HintonAdministrative, technical, or material support (i.e., reporting or organiz-ing data, constructing databases): K.A. Scarlett, C.V. HintonStudy supervision: K.A. Scarlett, E.Z. White, C.V. Hinton

AcknowledgmentsThis work was supported by grant funding to C.V. Hinton from NIH/

NIGMS (R01GM106020). Training support for students was provided, inpart, by NIH/NIGMS 2R25GM060414 and NIH/NIHMD 4P20MD002285;confocal analyses was funded by Research Centers in Minority InstitutionsProgram (RCMI)/NIMHD (5G12MD007590).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 3, 2017; revised July 12, 2017; acceptedDecember 20, 2017;published first January 12, 2018.

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