Bone Cell-autonomous Contribution of Type 2 Cannabinoid

Bone Cell-autonomous Contribution of Type 2 CannabinoidReceptor to Breast Cancer-induced Osteolysis*

Received for publication, March 9, 2015, and in revised form, July 20, 2015 Published, JBC Papers in Press, July 20, 2015, DOI 10.1074/jbc.M115.649608

Antonia Sophocleous‡§1, Silvia Marino‡¶1, John G. Logan‡§, Patrick Mollat�, Stuart H. Ralston§, and Aymen I. Idris‡¶2

From the ¶Academic Unit of Bone Biology, Mellanby Centre for Bone Research, Department of Human Metabolism, Medical School,Beech Hill Road, Sheffield S10 2RX, United Kingdom, ‡Bone and Cancer Group, Edinburgh Cancer Research Centre, Western GeneralHospital, University of Edinburgh, Edinburgh EH4 2XR, United Kingdom, §Rheumatology and Bone Diseases Unit, Centre forGenomic and Experimental Medicine, MRC Institute of Genetics and Molecular Medicine, Western General Hospital, University ofEdinburgh, Edinburgh EH4 2XU, United Kingdom, and �Galapagos SASU, 102 Avenue Gaston Roussel, 93230 Romainville, France

Background: CB2 is implicated in bone remodeling and tumor growth.Results: CB2 activation enhances breast cancer-induced bone cell activity and osteolysis via the PI3K/AKT pathway.Conclusion: CB2-selective antagonism has potential efficacy in cancer-associated bone disease.Significance: CB2 activation by phytocannabinoids might be detrimental in breast cancer patients with advancedmalignancy.

The cannabinoid type 2 receptor (CB2) has previously beenimplicated as a regulator of tumor growth, bone remodeling,and bone pain. However, very little is known about the role ofthe skeletal CB2 receptor in the regulation of osteoblasts andosteoclasts changes associated with breast cancer. Here wefound that the CB2-selective agonists HU308 and JWH133reduced the viability of a variety of parental and bone-tropichuman and mouse breast cancer cells at high micromolar con-centrations. Under conditions in which these ligands are used atthe nanomolar range, HU308 and JWH133 enhanced humanand mouse breast cancer cell-induced osteoclastogenesis andexacerbated osteolysis, and these effects were attenuated in cul-tures obtained from CB2-deficient mice or in the presence of aCB2 receptor blocker. HU308 and JWH133 had no effects onosteoblast growth or differentiation in the presence of condi-tioned medium from breast cancer cells, but under these cir-cumstances both agents enhanced parathyroid hormone-in-duced osteoblast differentiation and the ability to supportosteoclast formation. Mechanistic studies in osteoclast precur-sors and osteoblasts showed that JWH133 and HU308 inducedPI3K/AKT activity in a CB2-dependent manner, and theseeffects were enhanced in the presence of osteolytic and osteo-blastic factors such as RANKL (receptor activator of NF�Bligand) and parathyroid hormone. When combined with pub-lished work, these findings suggest that breast cancer andbone cells exhibit differential responses to treatment withCB2 ligands depending upon cell type and concentrationused. We, therefore, conclude that both CB2-selective activa-

tion and antagonism have potential efficacy in cancer-associ-ated bone disease, but further studies are warranted andongoing.

The endocannabinoid system comprises two known recep-tors (CB13 and CB2), a family of endogenous ligands andmolecular machinery for ligand synthesis, transport, and inac-tivation (1). CB1 and CB2 receptors belong to the G protein-coupled receptor superfamily that exhibits 44% homology atthe protein level (2) and shares a number of common signaltransduction pathways, including adenylyl cyclase (3), extracel-lular signal-regulated (ERK) kinases (p42/p44 MAPK) (4, 5),and the phosphatidylinositol 3-kinase/AKT (PI3/AKT) path-way (6). The CB1 receptors are highly expressed in the centralnervous system, whereas CB2 is predominately found in theimmune system and a number of other peripheral tissues (1).

�9-Tetrahydrocannabinol, the main psychotropic constitu-ent of cannabis (1), and various synthetic cannabinoid receptorligands have been extensively investigated as potential treat-ments for cancer with varying results. Depending on the ligandsused, cell lines studied, and disease models employed, cannabi-noid receptor ligands have been shown to exert both stimula-tory and inhibitory effects on cancer cell proliferation andtumor progression (7–16). In general terms, however, CB1 andCB2 receptor agonists have been found to have inhibitoryeffects on tumor cell growth, whereas antagonists and inverseagonists have been found to have stimulatory effects (7–16).Moreover, clinical studies have shown that cannabinoid recep-tor agonists exert analgesic and muscle relaxant properties inpatients with metastatic pain (7–18). The cannabinoid-baseddrug Sativex�, a plant extract that contains various plant-de-rived cannabinoids, is approved in some countries for the treat-

* This work was supported in part by an European Calcified Tissue Interna-tional Society/AMGEN (ECTS/AMGEN) fellowship grant (to A. I.) and a grantfrom the Arthritis Research UK (17713). Dr. A. I. Idris and Prof. S. H. Ralstonare co-inventors on a patent claiming the use of cannabinoid receptorligands as treatments for bone disease. Patrick Mollat is an employee ofGalapagos SASU.

1 Both authors contributed equally to manuscript.2 To whom correspondence should be addressed: Academic Unit of Bone

Biology, Mellanby Centre for Bone Research, Dept. of Human Metabo-lism, Medical School, Beech Hill Road, Sheffield S10 2RX, UK. Tel.:441142713338; E-mail: [email protected].

3 The abbreviations used are: CB1, cannabinoid type 1 receptor; CB2, canna-binoid type 2 receptor; RANKL, receptor activator of NF�B ligand; M-CSF,macrophage colony stimulating factor; PTX, pertussis toxin; MDA-231,MDA-MB-231; PTH, parathyroid hormone; TRAcP, tartrate resistant acidphosphatase; �-MEM, minimum Eagle’s medium; OPG, Osteoprotegerin.

crossmarkTHE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 36, pp. 22049 –22060, September 4, 2015

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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ment of cancer-associated pain (for review, see Ref. 16),whereas the synthetic equivalents of �9-tetrahydrocannabinolMarinol� and Cesamet� have been prescribed for the treat-ment of nausea and vomiting associated with cancer chemo-therapy (for review, see Ref. 16).

Most recent interest has been focused on the potential role ofCB2-selective agonists in the treatment of malignant disease,because these agents (a) lack of adverse psychotropic effectsthat are associated with CB1-selective ligands (19), (b) exertanti-proliferative effects on different cancer cell lines (20, 21),(c) inhibit cancer-induced osteolysis and fractures (22), and (d)reduce bone pain in various preclinical models (22, 23). At thepresent time CB2 agonists are considered to exert these effectsby inhibiting tumor cell growth and by suppressing the releaseof cytokines and chemokines from cancer cells (24). However,the cellular and molecular mechanisms by which CB2 agonistsprotect against tumor-induced osteolysis remains incompletelyunderstood. Over recent years we and others have reported thatcannabinoid receptors and their ligands directly regulate bonecell activity and bone mass (25–32). This raises the possibilitythat the effects of cannabinoid receptor ligands on models ofcancer-associated osteolysis might be mediated, not only byeffects on tumor cells, but also by the effects on bone cells. Inthis study we have employed genetic and pharmacologicalapproaches to examine the mechanisms by which the skeletalCB2 pathway regulates osteolysis mediated by breast cancer.

Experimental Procedures

Materials and Methods—The cannabinoid receptor agonistsHU308, JWH133, and AM630 were purchased from Tocris Bio-sciences (Bristol, UK). Human MDA-MB-231 (MDA-231) andMCF7 and mouse 4T1 breast cancer cell lines were purchasedfrom ATCC (Manassas, VA). Tissue culture medium (�-MEMand DMEM) was obtained from Invitrogen. The PI3 kinaseinhibitor LY294002 was purchased from New England BiolabsLtd. (Hertfordshire, UK), and the inhibitor of Gi/o proteins per-tussis toxin (PTX) was purchased from Sigma. Primers forquantitative PCR were designed using the Roche Diagnosticswebsite and obtained from Invitrogen, and probes were pur-chased from Roche Diagnostics All primary antibodies werepurchased from Cell Signaling Biotechnology except rabbitanti-CB2 receptor that was purchased from Cayman Chemicaland anti-actin that was obtained from Sigma. Mouse macro-phage colony stimulating factor (M-CSF) was obtained fromR&D Systems (Abingdon, UK), and receptor activator of NF�Bligand (RANKL) was a gift from Patrick Mollat (GalapagosSASU) and was prepared as previously described (33).

Cancer Cell Lines and Conditioned Medium—Human MDA-231 and MCF-7 and mouse 4T1 breast cancer cells were cul-tured in standard DMEM (DMEM supplemented with 10%FCS, penicillin, and streptomycin). For studies involving con-ditioned medium, breast cancer cells were cultured in standardDMEM and allowed to grow to 80% confluence over a period of48 –72 h. Medium was removed and replaced with serum-freeDMEM, and then the cells were incubated for a further 24 h.Conditioned medium form these cultures was filtered (0.22 �M

filter diameter) and used fresh (10 –20% v/v).

RANKL and M-CSF Mouse Osteoclast Culture—Mouse oste-oclast cultures were generated from bone marrow macro-phages stimulated with M-CSF and RANKL as previouslydescribed (30). For studies involving conditioned medium, con-ditioned medium prepared as described above was added toosteoclast cultures at a concentration of (10%, v/v) in standard�-MEM supplemented with M-CSF (25 ng/ml) and RANKL(100 ng/ml). Cultures were then treated with vehicle or testcompounds for the desired period of time. At the end of theculture period cells were fixed in 4% formaldehyde, washedwith PBS, and stained for tartrate-resistant acid phosphatase(TRAcP) as described in Aitken et al. (34). TRAcP-positive cellswith more than three nuclei were counted as osteoclasts.

Bone Marrow/Breast Cancer Cell Co-culture—Bone marrowmacrophages or M-CSF-generated osteoclast precursors wereplated into 96-well plates (10 � 103 cells/well) in 150 �l ofstandard �-MEM supplemented with M-CSF (25 ng/ml) andRANKL (100 ng/ml) for 6 h before the addition of MDA-231,MCF-7, or 4T1 breast cancer cells (300 cells/well). Cultureswere then treated with vehicle or test compounds for thedesired period of time. At the end of the culture period cellswere fixed in 4% formaldehyde, washed with PBS, and stainedfor TRAcP. TRAcP-positive cells with more than three nucleiwere counted as osteoclasts.

Quantification of Resorption Area—RANKL and M-CSF-stimulated osteoclasts cultures generated as described abovewere plated on Corning� Osteo Assay Surface multiple wellplates (Corning). Human MDA-231 breast cancer cells (300cells/well) were added to the mature osteoclast culture in thepresence or absence of treatment. Adherent osteoclasts wereincubated in 50% bleach (Clorox-Ultra) for 10 min, and thenresorption pits were visualized by phase contrast microscopyusing an Olympus ScanR microscope. The area resorbed wasquantified by Image Analysis using ImageJ.

Osteoblast Cultures in the Presence of Conditioned Medium—Primary osteoblasts were isolated from the calvarial bones of2-day-old mice by sequential collagenase digestion as previ-ously described (35). Osteoblasts were maintained in �-MEMsupplemented with 10% FCS and left to adhere overnight. Con-ditioned medium was then added to the osteoblast cultures(10% v/v), and cells were treated with vehicle or test compoundsfor the desired period of time. At the end of the experiment,osteoblast cultures were used for RNA isolation or to determineosteoblast number and differentiation by AlamarBlue assay andalkaline phosphatase assay, respectively. Both assays were per-formed as previously described (36).

Bone Marrow Cell/Osteoblast Co-cultures in the Presence ofConditioned Medium—Bone marrow cell populations con-taining osteoclast precursors were isolated using the Ficoll-Hypaque density gradient centrifugation technique as de-scribed in Idris et al. (30). These cells were then seeded in platescontaining adherent primary osteoblasts in the presence ofconditioned medium (10% v/v) prepared as described above.Cultures were then treated with vehicle or test compounds forthe desired periods of time. At the end of the culture period cellswere fixed in 4% formaldehyde, washed with PBS, and stainedfor TRAcP. TRAcP-positive cells with more than three nucleiwere counted as osteoclasts.

Skeletal CB2 Receptor Modulates Osteolysis

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Human Breast Cancer Cell/Mouse Calvarial Co-culture—Neonatal mouse calvaria were isolated from 7-day-old mice,washed thoroughly in Hanks’ balanced salt solution, and incu-bated in standard �-MEM as described in Garret (37). Eachmouse calvaria was then divided into two halves along themedian sagittal suture. Each half was placed in organ culture onstainless steel rafts in 48-well plates containing standardmedium (see Fig. 5A) and treated for 7 days with vehicle 0.1%DMSO or test compounds in the presence or absence of MDA-231 cells (10 � 103 cells/well). Osteolysis was assessed by mea-suring bone volume using microcomputed tomography (Sky-scan 1172 scanner, Skyscan, Belgium) at a resolution of 5 �m.Cancer cells from MDA-231/mouse calvarial organ cultureswere washed three times with ice-cold PBS and lysed, andsupernatant was collected. Total protein (50 �g) was resolvedon polyacrylamide gels, transferred onto PVDF membranes(Bio-Rad) and immunoblotted with human-cleaved and totalCaspase-3 antibodies (Santa Cruz Biotechnology) according tothe manufacturer’s instructions.

Western Blotting—Western blot analysis was used to detectprotein expression and activity in cultured cells. Cells wereseeded in 12-well plates and maintained in standard mediumuntil confluent. Before stimulation with test agents or vehicle,cells were incubated in serum-free medium for 60 min. Testagents or vehicle were prepared in serum-free medium andwere then added for the desired period of time. Cells were thenscraped, supernatant was collected, and protein concentrationwas determined as previously described (31). Total protein (50�g) was resolved on polyacrylamide gels, transferred ontoPVDF membranes (Bio-Rad), and immunoblotted with anti-bodies according to manufacturer’s instructions. Immunocom-plexes with primary and secondary antibodies were visualizedusing a chemiluminescent detection system (Fisher) on aSyngene Genegnome bioimaging system (Fisher) (31). Levels ofphosphorylated (or modified) proteins were normalized to totalprotein, and changes were expressed as the percentage ofcontrol.

Quantitative PCR—Gene expression was detected usingquantitative PCR. Total RNA was isolated, and complementaryDNA was generated as previously described (26). Primers weredesigned using the Ensembl genome browser and Roche Diag-nostics website for amplification of: mouse TRAcP (forwardprimer, 5�-CGTCTCTGCACAGATTGCAT-3�; reverse prim-er, 5�-AAGCGCAAACGGTAGTAAGG-3�, product size 75bp); mouse cathepsin K receptor (forward primer, 5�-CGAAA-AGAGCCTAGCGAACA-3�; reverse primer, 5�-TGGGTAG-CAGCAGAAACTTG-3�, product size 67 bp); mouse calcit-onin receptor (forward primer, 5�-GGTTCCTTCTCGTGAA-CAGGT-3�; reverse primer 5�-GCCTGAAGAACTGGAGT-TGG-3�, product size 70 bp); mouse OPG (forward primer, 5�-atgaacaagtggctgtgctg-3�; reverse primer, 5�-cagtttctgggtcataat-gcaa-3�); mouse RANKL (forward primer, 5�-tgaagacacactacc-tgactcctg-3�; reverse primer, 5�-ccacaatgtgttgcagttcc-3�);human GAPDH (forward primer, 5�-agccacatcgctcagacac-3�;reverse primer, 5�-gcccaatacgaccaaatcc-3�). The PCR protocolwas as follows: denaturation at 95 °C for 10 min followed by 35cycles of denaturation at 95 °C for 15 s and annealing at 60 °Cfor 30 s followed by extension at 72 °C for 15 s. Levels of gene

expression were expressed as copy number per microgram oftotal RNA and GAPDH was used for normalization.

Statistical Analysis—Comparison between groups was doneby analysis of variance followed by Dunnett’s post test usingSPSS for Windows Version 11. A p value of 0.05 or below wasconsidered statistically significant. The half-maximal inhibi-tory concentration (IC50) values were calculated usingGraphPad Prism 4 for windows. Data are the averages of threeindependent experiments, and reported error bars are S.D.unless stated otherwise.

Results

Effects of CB2 Receptor Agonists on Growth of Breast CancerCell Lines and Bone Cells—The CB2-selective agonists HU308and JWH133 inhibited growth of parental and bone-tropicbreast cancer cell lines MDA-231 and 4T1 and parental MCF7cells in the low micromolar range with half-maximal inhibitoryeffects (IC50) between the 5–10 �M range (Table 1). In contrast,no significant inhibitory effects on growth of osteoblasts orbone marrow-derived cells were observed at concentrations of�10 �M (Table 1).

CB2 Receptor Activation Stimulates Osteoblasts Support forOsteoclastogenesis—It is known that osteoblast-like cells sup-port osteoclastogenesis through secretion of various cytokinesincluding M-CSF and RANKL (38). In view of this, we exam-ined the effects of CB2 agonists on osteoclastogenesis in bonemarrow/osteoblast co-cultures exposed to conditionedmedium from a variety of human and mouse breast cancer cells(Fig. 1A). Conditioned medium from human MDA-231, humanMCF7, and mouse 4T1 breast cancer cells stimulated osteoclastformation in bone marrow cell/osteoblast co-cultures com-pared with control cultures (Fig. 1B). Moreover, both JWH133and HU308 at concentrations of 0.1–1 �M further enhanced thebreast cancer-induced osteoclast formation in these cultures(Fig. 1, B and C). This stimulatory effect of both ligands wasassociated with an increase in the RANKL/OPG ratio (50 �5.5% increase with JWH133, p � 0.05; 100 � 6.5% increase withHU308, p � 0.05) (Fig. 1, D–F). However, neither of the CB2receptor-selective ligands had an effect on osteoblast prolifera-tion (Fig. 1G). Together, these data indicate that one mecha-nism by which CB2 receptor activation in osteoblasts promotes

TABLE 1Effects of CB2 receptor selective agonists (half-maximal inhibitoryconcentration, IC50) on growth of breast cancer and bone cells in vitroCell numbers were measured after 48 h of continuous exposure to test agents. Cellviability was measured after 48 h of continuous exposure. Viability assay and calcu-lation of half-maximal inhibitory concentrations (IC50) have been performed asdescribed under “Materials and Methods.” Values are expressed as the means � S.D.and are obtained from five independent experiments. BT denotes bone tropic, andPre-osteoclasts refers to M-CSF-generated osteoclast precursors.

IC50

Cells JWH133 HU308

�M �M

MDA-231 9.87 � 1.1 9.3 � 1.5MDA-231-BT 7.24 � 2.1 6.11 � 1.6MCF7 8.71 � 0.9 5.83 � 1.14T1 8.87 � 1.6 8.30 � 1.74T1-BT 6.33 � 0.8 5.51 � 2.1Pre-osteoclasts �10 �10Osteoblasts �10 �10




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breast cancer-induced osteoclastogenesis is by increasing theRANKL/OPG ratio.

CB2 Receptor Agonists Enhance Breast Cancer-induced Oste-oclast Formation and Bone Resorption—To examine the directeffects of CB2 agonists on osteoclasts and their M-CSF- gener-

ated precursors, we went on to test the effects of JWH133 andHU308 on osteoclast formation in M-CSF- and RANKL-stim-ulated bone marrow cultures in the presence or absence of con-ditioned medium from a variety of human and mouse breastcancer cells (Fig. 2A). Pretreatment of M-CSF generated oste-

FIGURE 1. The CB2 receptor-selective agonists JWH133 and HU308 enhance osteoblasts support for osteoclastogenesis in a bone metastatic setting.A, experimental flow and timeline of the effects of conditioned medium on osteoblast differentiation and osteoblast support for osteoclastogenesis. CM,conditioned medium; BMC, bone marrow cells. B, number of osteoclasts in mouse bone marrow cell/osteoblast co-cultures in the presence or absence of theCB2-selective agonists JWH133 and HU308 (0.1–1 �M) after exposure to conditioned medium from human MDA-231 and MCF7 and mouse 4T1 breast cancercells (10% v/v). The average number of TRAcP positive multinucleated osteoclasts with three or more nuclei in vehicle treated control cultures are as follows:osteoblast/BMC, 27 � 6; osteoblast/BMC/MDA-231, 46 � 3; osteoblast/BMC/MCF7, 38 � 4; osteoblast/BMC/4T1, 45 � 4. C, representative photomicrographsof multinucleated TRAcP-positive osteoclasts from the experiment described in panel B. D–F, expression of RANKL (D) and OPG (E) as the percentage of GAPDHand RANKL/OPG ratio (F) in mouse calvarial osteoblasts in the presence or absence of the CB2-selective agonists JWH133 and HU308 (1 �M) after exposure toconditioned medium from human MDA-231 breast cancer cells (20% v/v). G, cell number in mouse calvarial osteoblasts in the presence or absence of theCB2-selective agonists JWH133 and HU308 (0.1–1 �M) after exposure to conditioned medium from human MDA-231 breast cancer cells (10% v/v). AU,absorbance units. *, p � 0.05 from vehicle control; �, p � 0.05 from vehicle in the presence of conditioned medium from MDA-231 breast cancer cells.




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oclast precursors with the CB2 receptor-selective agonistsJWH133 and HU308 at concentrations between 0.1 and 1 �M

for 1 h before stimulation with RANKL (100 ng/ml) enhanced

the effects of M-CSF and RANKL on osteoclast formation (Fig.2, B and C) and in the same cultures further enhanced the oste-oclastogenic effects of conditioned medium from human

FIGURE 2. The CB2 receptor-selective agonists JWH133 and HU308 enhance breast cancer-induced osteoclast formation and bone resorption. A,experimental flow and timeline of the effects of CB2 receptor-selective agonists on conditioned medium-induced osteoclast formation, survival, and activity.BMC, bone marrow cells. B, number of osteoclasts in bone marrow cultures stimulated with M-CSF (25 ng/ml) and RANKL (100 ng/ml) after exposure toconditioned media from human MDA-231 and MCF7 and mouse 4T1 breast cancer cells (10% v/v) in the presence or absence of the CB2-selective agonistsJWH133 and HU308 (0.1–1 �M). The average numbers of TRAcP-positive multinucleated osteoclasts with three or more nuclei in vehicle-treated controlcultures are as follows: BMC, 25 � 2; BMC/MDA-231, 45 � 9; BMC/MCF7, 49 � 7; BMC/4T1, 61 � 3. C, representative photomicrographs of multinucleatedTRAcP-positive osteoclasts from the experiment described in panel B. D, expression of the osteoclast-specific genes TRAcP, calcitonin receptor, and cathepsinK in bone marrow cultures stimulated with M-CSF (25 ng/ml) and RANKL (100 ng/ml) after exposure to conditioned medium from human MDA-231 breastcancer cells (20% v/v) in the presence or absence of the CB2 receptor-selective agonists JWH133 and HU308 (1 �M). E, resorbed area bone marrow culturesstimulated with M-CSF (25 ng/ml) and RANKL (100 ng/ml) after exposure to conditioned medium from human MDA-231 breast cancer cells (20% v/v) in thepresence or absence of the CB2-selective agonists JWH133 and HU308 (1 �M). F, number of mature osteoclasts after exposure to conditioned medium fromhuman MDA-231 breast cancer cells (10% v/v) in the presence or absence of the CB2-selective agonists JWH133 and HU308 (0.1–1 �M) after withdrawal ofRANKL (100 ng/ml) or M-CSF (25 ng/ml). *, p � 0.05 from vehicle control; �, p � 0.05 from vehicle in the presence of conditioned medium from MDA-231 breastcancer cells. CM, conditioned medium.




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MDA-231, human MCF7, and mouse 4T1 breast cancer cells(Fig. 2, B and C). In keeping with this, expression of osteoclast-specific genes, such as TRAcP, calcitonin receptor, and cathep-sin K, were increased by JWH133 and HU308 in these cultures(Fig. 2D). Bone resorption was also significantly enhanced byJWH133 (35% � 14 increase) and HU308 (41% � 1.6 increase)in similar cultures using conditioned medium from humanMDA-231 (Fig. 2E). Although breast cancer cell-conditionedmedium and the CB2-selective agonists enhanced the oste-oclastogenic effects of M-CSF and RANKL, these factors aloneand in combination were unable to support osteoclast forma-tion in the absence of RANKL and M-CSF (Fig. 2F). Further-more, the addition of JWH133 and HU308 to MDA-231-con-ditioned medium enhanced M-CSF- and RANKL-inducedosteoclast formation in wild type cultures, but no effect wasobserved in bone marrow cultures from mice lacking CB2receptors (CB2/ mice) (see Fig. 4, A and B). In accordancewith this, pretreatment with the CB2-selective antagonist/in-verse agonist AM630 or the Gi/o PTX for 1 h before addingHU308 and JWH133 in wild type cultures significantly inhib-ited osteoclast formation (see Fig. 4A). Collectively, theseresults suggest that JWH133 and HU308 enhance breast can-cer-induced osteoclast formation by CB2 receptors in a PTX-sensitive manner.

Role of the Akt Pathway in CB2-induced Osteoclastogenesis—The PI3/AKT signaling transduction pathway is known to playa role in osteoclastogenesis downstream of various receptorsincluding RANK and M-CSF in osteoclasts and the parathyroidhormone (PTH) receptor in osteoblasts (39). To determinewhether CB2 agonists promote AKT phosphorylation in thebone marrow microenvironment, we tested the effects of breastcancer-conditioned medium and CB2 agonists on phosphati-dylinositol 3-kinase/AKT (PI3K/AKT) phosphorylation. First,we show that treatment with the CB2-selective agonistsJWH133 or HU308 significantly induced the phosphorylationof AKT at serine 473, but not threonine 308, in M-CSF-stimu-lated bone marrow cells within 15 min (Fig. 3, A and B). More-over, both CB2 receptor-selective agonists enhanced RANKL-induced AKT phosphorylation at serine 473 (Fig. 3, E and F). Ofnote, we have not detected any changes in the levels of phos-phorylated or total AKT in these cultures for up to 24 h posttreatment (Fig. 3, C and D). The addition of conditionedmedium from MDA-231 cells enhanced AKT phosphorylationin RANKL and M-CSF-stimulated bone marrow cells, and thiswas further increased by JWH133 and HU308 (Fig. 4, C and D).This stimulatory effect was abolished by preincubation with thePI3K/AKT inhibitor LY294002, the Gi/o inhibitor PTX, and theCB2-selective antagonist/inverse agonist AM630 (Fig. 4, C andD). Previously, we reported that PTH, a potent stimulator ofosteoblast differentiation and AKT activity (42, 43), enhancesalkaline phosphatase activity in wild type calvarial osteoblastsbut not in cultures from mice deficient in CB2 receptors(CB2/ mice) (31). Here, we examined the effects of PTH onosteoblast cultures exposed to conditioned medium fromMDA-231 breast cancer cells. This showed that PTH stimu-lated alkaline phosphatase in osteoblast cultures and that theCB2 agonists JHW133 and HU308 further increased alkalinephosphatase levels (Fig. 5A). Similarly, PTH stimulated oste-

oclast formation in mouse osteoblasts/bone marrow cell cul-tures in the presence of MDA-231 cell-conditioned medium(Fig. 5, B and C). Osteoclast formation was further increased bythe combination of PTH and the CB2 agonists JWH133 andHU308 (Fig. 5, B and C). In cultures of calvarial osteoblasts,however, JWH133 and HU308 enhanced PTH-induced phos-phorylation of AKT at threonine 308 (Fig. 5, D and E). Thesedata together demonstrate that breast cancer cell conditionedmedium stimulates AKT phosphorylation in bone marrow cellsand that this is increased by PTH and further increased by CB2agonists. This illustrates that another mechanism by which theCB2 pathway enhances osteoclastogenesis is through AKTphosphorylation.

Involvement of the CB2 Receptor in Focal Osteolysis Is Medi-ated by Breast Cancer Cells—Next, we investigated the role ofCB2 receptor activation on breast cancer-induced osteolysis inan organ culture model by using MDA-231 human breast can-cer cells/mouse calvarial organ culture (Fig. 6A). The additionof MDA-231 cells to the organ cultures from wild type micecaused a dramatic decrease in calvarial bone volume, reaching73% � 2.9 bone loss (p � 0.01) over a 7-day-culture period (Fig.6B). Treatment with the CB2 receptor-selective agonistsJWH133 and HU308 increased osteolysis even further to 87% �2.7 (p � 0.05) and 92% � 3.1 (p � 0.05), respectively (Fig. 6, Band C). However, osteolysis was reduced after treatment withthe CB2-elective antagonist/inverse agonist AM630 and inorgan co-cultures from CB2/ mice (Fig. 6B). MDA-231 cellsfrom these organ co-cultures were not affected by JWH133 orHU308 treatment either in terms or viability (Fig. 6D) or sur-vival, indicated by the lack of caspase-3 activation (Fig. 6E).These results demonstrate that the CB2 receptor in bone cellsplays a key role in regulating local osteolysis mediated by breastcancer cells.

Discussion

There has been an increasing interest in the potential use ofcannabinoid ligands for cancer treatment. The present studiesillustrate that the CB2 pathway plays a role in enhancing oste-oclast activation mediated by tumor cell conditioned mediumas well as exerting an inhibitory effect on growth of breast can-cer cell lines. We also provide evidence for the first time show-ing that the stimulatory effects of CB2 are mediated not only bythe effects on tumor cells but also by CB2 receptors expressedby bone cells. Evidence for this comes from the observation thatCB2 agonists had no stimulatory effects on osteoclast forma-tion in bone cell cultures from CB2-deficient mice and that thestimulatory effects of breast cancer cells on local osteolysis wereattenuated in calvarial explant cultures from CB2-deficientmice.

We and others have previously shown that activation of can-nabinoid receptors with agonists stimulate osteoclast forma-tion, whereas treatment with cannabinoid receptor antago-nists/inverse agonists inhibit osteoclastogenesis in vitro andovariectomy-induced bone loss in vivo (25, 30, 40). Ofek et al.(29), on the other hand, proposed that the CB2-seletive agonistHU308 inhibits osteoclast formation in vitro and in vivo. In thepresent study not only have we confirmed that CB2 receptoractivation with the non-psychoactive agonists JWH133 and




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HU308 significantly stimulates osteoclastogenesis in bone mar-row cultures, but we also demonstrated that in the presence ofconditioned medium from different breast cancer cell lines aswell as breast cancer cells per se, these CB2-selective agonistssignificantly enhance osteoclast formation and osteoclasticresorption. These stimulatory effects were abolished when

bone marrow cultures were prepared from mice deficient inCB2 receptors. This was mirrored in a pharmacologic inactiva-tion context, when cultures were treated with the CB2-selectiveantagonist/inverse agonist AM630, pointing to the fact that anyform of inactivation of the CB2 receptors suppresses osteoclas-togenesis in a metastatic bone environment.

FIGURE 3. JWH133 and HU308 induce PI3K/AKT activation in osteoclasts in the presence and absence of RANKL. A, Western blot analysis of AKTphosphorylation in M-CSF-generated bone marrow cells exposed to the CB2-selective agonists JWH133 or HU308 (1 �M) for 15 min. B, quantification of AKTphosphorylation from the experiment described in panel A. C, Western blot analysis of AKT phosphorylation in M-CSF-generated bone marrow cells exposedto the CB2-selective agonists JWH133 or HU308 (1 �M) for 24 h. D, quantification of AKT phosphorylation from the experiment described in panel C. E, Westernblot analysis of AKT phosphorylation in bone marrow cultures stimulated with M-CSF after exposure to RANKL in the presence or absence of the CB2-selectiveagonists JWH133 and HU308 (1 �M). F, quantification of AKT phosphorylation from the experiment described in panel E. *, p � 0.05 from vehicle control; �, p �0.05 from WT vehicle in the presence of conditioned medium from MDA-231 breast cancer cells.




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Previously, we showed that alkaline phosphatase activity, amarker of osteoblast differentiation, is enhanced by PTH inwild type calvarial osteoblasts but not in cultures from CB2/

mice (31). Here, PTH-induced alkaline phosphatase activity inthe presence of conditioned media was further enhanced aftertreatment with JWH133 and HU308, supporting the fact thatactivation of CB2 receptors increases osteoblast differentiationin a metastatic setting. Moreover, CB2 activation stimulatesosteoblast support for osteoclastogenesis by enhancing theRANKL/OPG ratio in the presence of conditioned media frombreast cancer cells. It is well established that breast cancer and

bone cells secrete various factors that increase RANKL andinhibit OPG expression by osteoblasts (41). Although the sig-naling mechanisms by which cannabinoid receptor ligandsaffect bone cell differentiation and function are thus far rela-tively unknown, the current study suggests that in M-CSF-gen-erated osteoclast precursors and osteoblasts, cannabinoidreceptor agonists enhance phosphorylation of AKT, suggestingthat this may be one mechanism by which CB2 activation con-tributes to osteoclastic and osteoblastic effects associated withosteolysis. It is worthwhile to mention that neither HU308 norJWH133 had any significant effects on the activation and phos-

FIGURE 4. JWH133 and HU308 enhance breast cancer-induced osteoclast formation via CB2 receptors in a PTX sensitive manner and a PI3K/AKTmechanism. A, number of osteoclasts in bone marrow cultures stimulated with mouse M-CSF and RANKL from wild type and CB2-deficient mice (CB2/) aftertreatment with the CB2-selective agonists JWH133 and HU308 (1 �M) and exposure to conditioned medium from human MDA-231 breast cancer cells (10% v/v)in the presence or absence of the CB2-selective antagonist/inverse agonist AM630 (3 �M) or the Gi/o inhibitor PTX (10 �M). The average number of TRAcP-positive multinucleated osteoclasts with three or more nuclei in wild type (WT) control cultures is 35 � 6. B, representative photomicrographs of multinucle-ated TRAcP-positive osteoclasts from the experiment described in panel A. C, Western blot analysis of AKT phosphorylation in bone marrow cultures stimulatedwith M-CSF and RANKL, exposed to conditioned medium from MDA-231 breast cancer cells (20% v/v), and then treated with the CB2-selective agonists JWH133and HU308 (1 �M) in the presence or absence of the CB2-selective antagonist/inverse agonist AM630 (1 �M), PI3K/AKT inhibitor LY294002 (10 �M), or PTX (10�M). D, quantification of phosphorylated AKT at serine 473 as percentage of total AKT from the experiment described in panel C. *, p � 0.05 from vehiclecontrol; �, p � 0.05 from WT vehicle in the presence of conditioned medium from MDA-231 breast cancer cells; $, p � 0.05 from AM630-, LY294002-, orPTH-treated. CM, conditioned medium; M, marker.




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FIGURE 5. The CB2 receptor-selective agonists JWH133 and HU308 enhance PTH induced osteoblasts and osteoclast changes in a bone metastaticsetting. A, alkaline phosphatase activity in mouse calvarial osteoblasts pre-exposed to conditioned medium from human MDA-231 breast cancer cells (10%v/v) then treated with the CB2-selective agonists JWH133 and HU308 (1 �M) in the presence of PTH (100 ng/ml). B, number of osteoclasts in mouse osteoblasts/bone marrow cell cultures pre-exposed to conditioned medium from human MDA-231 breast cancer cells (10% v/v) and then treated with the CB2-selectiveagonists JWH133 and HU308 (1 �M) in the presence of PTH (100 ng/ml). The average number of TRAcP-positive multinucleated osteoclasts with three or morenuclei in WT control cultures was 43 � 7. C, representative photomicrographs of multinucleated TRAcP-positive osteoclasts from the experiment described inpanel B. D, Western blot analysis of AKT phosphorylation in mouse calvarial osteoblast cultures pre-exposed to conditioned medium from human MDA-231breast cancer cells (20% v/v) and then treated with the CB2-selective agonists JWH133 and HU308 (1 �M) in the presence of PTH (100 ng/ml). E, quantificationof phosphorylated AKT at threonine 308 as the percentage of total AKT from the experiment described in panel D. *, p � 0.05 from vehicle control; �, p � 0.05from PTH in the presence of conditioned medium from MDA-231 breast cancer cells. CM, conditioned medium.




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phorylation of a number of key signaling proteins and tran-scription factors downstream of RANK receptor, namely IKK�and -�, I�B, p38, ERK1/2, MEK1/2, JNK1/2, p65NF�B,NFATc1, and cFOS (data not shown). Therefore, future studiesare still needed to evaluate the mechanism by which CB2 recep-

tor signaling in bone cells influences different aspects of AKTactivity such as posttranslational modifications.

Previous investigators showed that pharmacological target-ing of CB2 receptors with non-psychoactive CB2-selective ago-nists reduced cancer-induced bone loss and bone fracture (22,

FIGURE 6. JWH133 and HU308 exacerbate breast cancer-induced osteolysis via CB2 receptor activation. A, graphic representation of human breastcancer cell/mouse calvaria organ co-culture system. B, total bone volume loss in mouse calvaria bone from wild type and CB2-deficient mice (CB2/)co-cultured with human MDA-231 breast cancer cells (10 � 103 cells/well) in the presence or absence of the CB2-selective agonists JWH133 and HU308 (1 �M)or the CB2-selective antagonist/inverse agonist AM630 (1 �M). C, representative photomicrographs of microcomputed tomography scans and TRAcP- andaniline-stained histological sections of mouse calvarial bone from the experiment described in panel B, showing osteolysis. D, cell number of human MDA-231cells in breast cancer cell/mouse calvaria organ co-culture system treated with the CB2-selective agonists JWH133 and HU308 (1 �M) for 7 days. E, Western blotanalysis of total and cleaved Caspase-3 as well as actin in human MDA-231 cells in breast cancer cell/mouse calvaria organ co-culture system treated with theCB2-selective agonists JWH133 and HU308 (1 �M) for 7 days. �, p � 0.05 from vehicle in the presence of MDA-231 breast cancer cells; $, p � 0.05 from WTcultures treated with JWH133 and HU308 alone in the presence of MDA-231 breast cancer cells.




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24). However, these effects were attributed on the anti-prolif-erative actions of the CB2-selective agonists on cancer cellsrather than on an inhibitory effect directed on osteoclasts spe-cifically. Although we cannot exclude the possibility that theCB2 agonists studied here have inhibitory effects on tumorgrowth, the concentrations of these agents required to inhibitgrowth of tumor cells were three times higher than those thatwere shown here to enhance osteolysis that resulted fromtumor conditioned medium. In addition, the experiments incultured calvarial explants and cultures from CB2-deficientmice demonstrate clearly that at least part of the effect wasmediated by the cannabinoid pathway in bone cells rather thanthe tumor. It should also be noted that the CB2-selective agentsJWH133 and HU308 appeared to exacerbate osteolysis in cal-varial organ co-cultures from CB2-deficient mice. Although wecannot exclude the possibility that CB2 independent effectsexerted by the HU308 and JWH133 may have contributed tothe effects that we observed, the present study shows that CB2exerts bone cell-autonomous effects on differentiation of oste-oclasts and exerts direct effects on osteolysis. Further studiesusing cultured calvarial explants and cultures from cell-specificor neuron-specific inactivation of CB2 will be required toaddress the relative importance of signaling by CB1 and/orother related receptors to the regulation of osteolysis describedin this study.

In conclusion, our present findings suggest that inhibition ofCB2 receptor signaling in the bone microenvironment mayhave a potential role in protecting the skeleton from the oste-olysis associated with breast cancer. When combined with pre-vious studies, these findings suggest that the skeletal CB2receptor exhibits differential responses to treatment with CB2ligands and raises the possibility that both CB2-selective acti-vation and antagonism have potential efficacy in cancer-asso-ciated bone disease. The potential use of CB2 receptor agonistsin cases of breast cancer with bone metastases needs to be care-fully explored so that any treatment regime would take intoconsideration and exploit both their cell-autonomous effects inthe bone microenvironment and their direct effects on tumor.For that, further in vivo studies are warranted and ongoing.

Author Contributions—A. S. and S. M. acquired, analyzed, andinterpreted the data, J. G. L. acquired the data, P. M. is responsiblefor the conception and donation of material, S. H. R. revised the arti-cle and interpreted the data, and A. I. I. made substantial contribu-tions to conception and design, acquisition, analysis, and interpreta-tion of data and writing and revising the article.

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and Aymen I. IdrisAntonia Sophocleous, Silvia Marino, John G. Logan, Patrick Mollat, Stuart H. Ralston

Cancer-induced OsteolysisBone Cell-autonomous Contribution of Type 2 Cannabinoid Receptor to Breast

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