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

Antonia Sophocleous; Silvia Marino; John G Logan; Patrick Mollat; Stuart H Ralston; Aymen I Idris

doi:10.1074/jbc.M115.649608

. 2015 Jul 20;290(36):22049–22060. doi: 10.1074/jbc.M115.649608

Bone Cell-autonomous Contribution of Type 2 Cannabinoid Receptor to Breast Cancer-induced Osteolysis^*

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

PMCID: PMC4571957 PMID: 26195631

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 advanced malignancy.

Keywords: Akt PKB, cannabinoid receptor, G protein-coupled receptor (GPCR), osteoblast, osteoclast, bone, osteolysis

Abstract

The cannabinoid type 2 receptor (CB2) has previously been implicated as a regulator of tumor growth, bone remodeling, and bone pain. However, very little is known about the role of the skeletal CB2 receptor in the regulation of osteoblasts and osteoclasts changes associated with breast cancer. Here we found that the CB2-selective agonists HU308 and JWH133 reduced the viability of a variety of parental and bone-tropic human and mouse breast cancer cells at high micromolar concentrations. Under conditions in which these ligands are used at the nanomolar range, HU308 and JWH133 enhanced human and mouse breast cancer cell-induced osteoclastogenesis and exacerbated osteolysis, and these effects were attenuated in cultures obtained from CB2-deficient mice or in the presence of a CB2 receptor blocker. HU308 and JWH133 had no effects on osteoblast growth or differentiation in the presence of conditioned medium from breast cancer cells, but under these circumstances both agents enhanced parathyroid hormone-induced osteoblast differentiation and the ability to support osteoclast formation. Mechanistic studies in osteoclast precursors and osteoblasts showed that JWH133 and HU308 induced PI3K/AKT activity in a CB2-dependent manner, and these effects were enhanced in the presence of osteolytic and osteoblastic factors such as RANKL (receptor activator of NFκB ligand) and parathyroid hormone. When combined with published work, these findings suggest that breast cancer and bone cells exhibit differential responses to treatment with CB2 ligands depending upon cell type and concentration used. We, therefore, conclude that both CB2-selective activation and antagonism have potential efficacy in cancer-associated bone disease, but further studies are warranted and ongoing.

Introduction

The endocannabinoid system comprises two known receptors (CB1³ and CB2), a family of endogenous ligands and molecular machinery for ligand synthesis, transport, and inactivation (1). CB1 and CB2 receptors belong to the G protein-coupled receptor superfamily that exhibits 44% homology at the protein level (2) and shares a number of common signal transduction pathways, including adenylyl cyclase (3), extracellular signal-regulated (ERK) kinases (p42/p44 MAPK) (4, 5), and the phosphatidylinositol 3-kinase/AKT (PI3/AKT) pathway (6). The CB1 receptors are highly expressed in the central nervous system, whereas CB2 is predominately found in the immune system and a number of other peripheral tissues (1).

Δ⁹-Tetrahydrocannabinol, the main psychotropic constituent of cannabis (1), and various synthetic cannabinoid receptor ligands have been extensively investigated as potential treatments for cancer with varying results. Depending on the ligands used, cell lines studied, and disease models employed, cannabinoid receptor ligands have been shown to exert both stimulatory and inhibitory effects on cancer cell proliferation and tumor progression (7 –16). In general terms, however, CB1 and CB2 receptor agonists have been found to have inhibitory effects on tumor cell growth, whereas antagonists and inverse agonists have been found to have stimulatory effects (7 –16). Moreover, clinical studies have shown that cannabinoid receptor agonists exert analgesic and muscle relaxant properties in patients with metastatic pain (7 –18). The cannabinoid-based drug Sativex®, a plant extract that contains various plant-derived cannabinoids, is approved in some countries for the treatment of cancer-associated pain (for review, see Ref. 16), whereas the synthetic equivalents of Δ⁹-tetrahydrocannabinol Marinol® and Cesamet® have been prescribed for the treatment of nausea and vomiting associated with cancer chemotherapy (for review, see Ref. 16).

Most recent interest has been focused on the potential role of CB2-selective agonists in the treatment of malignant disease, because these agents (a) lack of adverse psychotropic effects that are associated with CB1-selective ligands (19), (b) exert anti-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 the present time CB2 agonists are considered to exert these effects by inhibiting tumor cell growth and by suppressing the release of cytokines and chemokines from cancer cells (24). However, the cellular and molecular mechanisms by which CB2 agonists protect against tumor-induced osteolysis remains incompletely understood. Over recent years we and others have reported that cannabinoid receptors and their ligands directly regulate bone cell activity and bone mass (25 –32). This raises the possibility that the effects of cannabinoid receptor ligands on models of cancer-associated osteolysis might be mediated, not only by effects on tumor cells, but also by the effects on bone cells. In this study we have employed genetic and pharmacological approaches to examine the mechanisms by which the skeletal CB2 pathway regulates osteolysis mediated by breast cancer.

Experimental Procedures

Materials and Methods

The cannabinoid receptor agonists HU308, JWH133, and AM630 were purchased from Tocris Biosciences (Bristol, UK). Human MDA-MB-231 (MDA-231) and MCF7 and mouse 4T1 breast cancer cell lines were purchased from ATCC (Manassas, VA). Tissue culture medium (α-MEM and DMEM) was obtained from Invitrogen. The PI3 kinase inhibitor LY294002 was purchased from New England Biolabs Ltd. (Hertfordshire, UK), and the inhibitor of G_i/o proteins pertussis toxin (PTX) was purchased from Sigma. Primers for quantitative PCR were designed using the Roche Diagnostics website and obtained from Invitrogen, and probes were purchased from Roche Diagnostics All primary antibodies were purchased from Cell Signaling Biotechnology except rabbit anti-CB2 receptor that was purchased from Cayman Chemical and anti-actin that was obtained from Sigma. Mouse macrophage colony stimulating factor (M-CSF) was obtained from R&D Systems (Abingdon, UK), and receptor activator of NFκB ligand (RANKL) was a gift from Patrick Mollat (Galapagos SASU) 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 cultured in standard DMEM (DMEM supplemented with 10% FCS, penicillin, and streptomycin). For studies involving conditioned medium, breast cancer cells were cultured in standard DMEM and allowed to grow to 80% confluence over a period of 48–72 h. Medium was removed and replaced with serum-free DMEM, and then the cells were incubated for a further 24 h. Conditioned medium form these cultures was filtered (0.22 μm filter diameter) and used fresh (10–20% v/v).

RANKL and M-CSF Mouse Osteoclast Culture

Mouse osteoclast cultures were generated from bone marrow macrophages stimulated with M-CSF and RANKL as previously described (30). For studies involving conditioned medium, conditioned medium prepared as described above was added to osteoclast cultures at a concentration of (10%, v/v) in standard α-MEM supplemented with M-CSF (25 ng/ml) and RANKL (100 ng/ml). Cultures were then treated with vehicle or test compounds for the desired period of time. At the end of the culture period cells were fixed in 4% formaldehyde, washed with PBS, and stained for tartrate-resistant acid phosphatase (TRAcP) as described in Aitken et al. (34). TRAcP-positive cells with more than three nuclei were counted as osteoclasts.

Bone Marrow/Breast Cancer Cell Co-culture

Bone marrow macrophages or M-CSF-generated osteoclast precursors were plated into 96-well plates (10 × 10³ cells/well) in 150 μl of standard α-MEM supplemented with M-CSF (25 ng/ml) and RANKL (100 ng/ml) for 6 h before the addition of MDA-231, MCF-7, or 4T1 breast cancer cells (300 cells/well). Cultures were then treated with vehicle or test compounds for the desired period of time. At the end of the culture period cells were fixed in 4% formaldehyde, washed with PBS, and stained for TRAcP. TRAcP-positive cells with more than three nuclei were counted as osteoclasts.

Quantification of Resorption Area

RANKL and M-CSF-stimulated osteoclasts cultures generated as described above were plated on Corning® Osteo Assay Surface multiple well plates (Corning). Human MDA-231 breast cancer cells (300 cells/well) were added to the mature osteoclast culture in the presence or absence of treatment. Adherent osteoclasts were incubated in 50% bleach (Clorox-Ultra) for 10 min, and then resorption pits were visualized by phase contrast microscopy using an Olympus ScanR microscope. The area resorbed was quantified by Image Analysis using ImageJ.

Osteoblast Cultures in the Presence of Conditioned Medium

Primary osteoblasts were isolated from the calvarial bones of 2-day-old mice by sequential collagenase digestion as previously described (35). Osteoblasts were maintained in α-MEM supplemented with 10% FCS and left to adhere overnight. Conditioned medium was then added to the osteoblast cultures (10% v/v), and cells were treated with vehicle or test compounds for the desired period of time. At the end of the experiment, osteoblast cultures were used for RNA isolation or to determine osteoblast number and differentiation by AlamarBlue assay and alkaline phosphatase assay, respectively. Both assays were performed as previously described (36).

Bone Marrow Cell/Osteoblast Co-cultures in the Presence of Conditioned Medium

Bone marrow cell populations containing osteoclast precursors were isolated using the Ficoll-Hypaque density gradient centrifugation technique as described in Idris et al. (30). These cells were then seeded in plates containing adherent primary osteoblasts in the presence of conditioned medium (10% v/v) prepared as described above. Cultures were then treated with vehicle or test compounds for the desired periods of time. At the end of the culture period cells were fixed in 4% formaldehyde, washed with PBS, and stained for TRAcP. TRAcP-positive cells with more than three nuclei were counted as osteoclasts.

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 incubated in standard α-MEM as described in Garret (37). Each mouse calvaria was then divided into two halves along the median sagittal suture. Each half was placed in organ culture on stainless steel rafts in 48-well plates containing standard medium (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 measuring bone volume using microcomputed tomography (Skyscan 1172 scanner, Skyscan, Belgium) at a resolution of 5 μm. Cancer cells from MDA-231/mouse calvarial organ cultures were washed three times with ice-cold PBS and lysed, and supernatant was collected. Total protein (50 μg) was resolved on polyacrylamide gels, transferred onto PVDF membranes (Bio-Rad) and immunoblotted with human-cleaved and total Caspase-3 antibodies (Santa Cruz Biotechnology) according to the manufacturer's instructions.

FIGURE 5. — **The CB2 receptor-selective agonists JWH133 and HU308 enhance PTH induced osteoblasts and osteoclast changes in a bone metastatic setting.** 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 μm) 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-selective agonists JWH133 and HU308 (1 μm) in the presence of PTH (100 ng/ml). The average number of TRAcP-positive multinucleated osteoclasts with three or more nuclei in WT control cultures was 43 ± 7. C, representative photomicrographs of multinucleated TRAcP-positive osteoclasts from the experiment described in *panel B. D*, Western blot analysis of AKT phosphorylation in mouse calvarial osteoblast cultures pre-exposed to conditioned medium from human MDA-231 breast cancer cells (20% v/v) and then treated with the CB2-selective agonists JWH133 and HU308 (1 μm) in the presence of PTH (100 ng/ml). E, quantification of 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 < 0.05 from PTH in the presence of conditioned medium from MDA-231 breast cancer cells. CM, conditioned medium.

Western Blotting

Western blot analysis was used to detect protein expression and activity in cultured cells. Cells were seeded in 12-well plates and maintained in standard medium until confluent. Before stimulation with test agents or vehicle, cells were incubated in serum-free medium for 60 min. Test agents or vehicle were prepared in serum-free medium and were then added for the desired period of time. Cells were then scraped, supernatant was collected, and protein concentration was determined as previously described (31). Total protein (50 μg) was resolved on polyacrylamide gels, transferred onto PVDF membranes (Bio-Rad), and immunoblotted with antibodies according to manufacturer's instructions. Immunocomplexes with primary and secondary antibodies were visualized using a chemiluminescent detection system (Fisher) on a Syngene Genegnome bioimaging system (Fisher) (31). Levels of phosphorylated (or modified) proteins were normalized to total protein, and changes were expressed as the percentage of control.

Quantitative PCR

Gene expression was detected using quantitative PCR. Total RNA was isolated, and complementary DNA was generated as previously described (26). Primers were designed using the Ensembl genome browser and Roche Diagnostics website for amplification of: mouse TRAcP (forward primer, 5′-CGTCTCTGCACAGATTGCAT-3′; reverse primer, 5′-AAGCGCAAACGGTAGTAAGG-3′, product size 75 bp); mouse cathepsin K receptor (forward primer, 5′-CGAAAAGAGCCTAGCGAACA-3′; reverse primer, 5′-TGGGTAGCAGCAGAAACTTG-3′, product size 67 bp); mouse calcitonin receptor (forward primer, 5′-GGTTCCTTCTCGTGAACAGGT-3′; reverse primer 5′-GCCTGAAGAACTGGAGTTGG-3′, product size 70 bp); mouse OPG (forward primer, 5′-atgaacaagtggctgtgctg-3′; reverse primer, 5′-cagtttctgggtcataatgcaa-3′); mouse RANKL (forward primer, 5′-tgaagacacactacctgactcctg-3′; reverse primer, 5′-ccacaatgtgttgcagttcc-3′); human GAPDH (forward primer, 5′-agccacatcgctcagacac-3′; reverse primer, 5′-gcccaatacgaccaaatcc-3′). The PCR protocol was as follows: denaturation at 95 °C for 10 min followed by 35 cycles of denaturation at 95 °C for 15 s and annealing at 60 °C for 30 s followed by extension at 72 °C for 15 s. Levels of gene expression were expressed as copy number per microgram of total RNA and GAPDH was used for normalization.

Statistical Analysis

Comparison between groups was done by analysis of variance followed by Dunnett's post test using SPSS for Windows Version 11. A p value of 0.05 or below was considered statistically significant. The half-maximal inhibitory concentration (IC₅₀) values were calculated using GraphPad Prism 4 for windows. Data are the averages of three independent experiments, and reported error bars are S.D. unless stated otherwise.

Results

Effects of CB2 Receptor Agonists on Growth of Breast Cancer Cell Lines and Bone Cells

The CB2-selective agonists HU308 and JWH133 inhibited growth of parental and bone-tropic breast cancer cell lines MDA-231 and 4T1 and parental MCF7 cells in the low micromolar range with half-maximal inhibitory effects (IC₅₀) between the 5–10 μm range (Table 1). In contrast, no significant inhibitory effects on growth of osteoblasts or bone marrow-derived cells were observed at concentrations of >10 μm (Table 1).

TABLE 1.

Effects of CB2 receptor selective agonists (half-maximal inhibitory concentration, IC₅₀) on growth of breast cancer and bone cells in vitro

Cell numbers were measured after 48 h of continuous exposure to test agents. Cell viability was measured after 48 h of continuous exposure. Viability assay and calculation of half-maximal inhibitory concentrations (IC₅₀) have been performed as described under “Materials and Methods.” Values are expressed as the means ± S.D. and are obtained from five independent experiments. BT denotes bone tropic, and Pre-osteoclasts refers to M-CSF-generated osteoclast precursors.

Cells	IC₅₀
Cells	JWH133	HU308
	μm	μm
MDA-231	9.87 ± 1.1	9.3 ± 1.5
MDA-231-BT	7.24 ± 2.1	6.11 ± 1.6
MCF7	8.71 ± 0.9	5.83 ± 1.1
4T1	8.87 ± 1.6	8.30 ± 1.7
4T1-BT	6.33 ± 0.8	5.51 ± 2.1
Pre-osteoclasts	>10	>10
Osteoblasts	>10	>10

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CB2 Receptor Activation Stimulates Osteoblasts Support for Osteoclastogenesis

It is known that osteoblast-like cells support osteoclastogenesis through secretion of various cytokines including M-CSF and RANKL (38). In view of this, we examined the effects of CB2 agonists on osteoclastogenesis in bone marrow/osteoblast co-cultures exposed to conditioned medium from a variety of human and mouse breast cancer cells (Fig. 1A). Conditioned medium from human MDA-231, human MCF7, and mouse 4T1 breast cancer cells stimulated osteoclast formation in bone marrow cell/osteoblast co-cultures compared with control cultures (Fig. 1B). Moreover, both JWH133 and HU308 at concentrations of 0.1–1 μm further enhanced the breast cancer-induced osteoclast formation in these cultures (Fig. 1, B and C). This stimulatory effect of both ligands was associated with an increase in the RANKL/OPG ratio (50 ± 5.5% increase with JWH133, p < 0.05; 100 ± 6.5% increase with HU308, p < 0.05) (Fig. 1, D–F). However, neither of the CB2 receptor-selective ligands had an effect on osteoblast proliferation (Fig. 1G). Together, these data indicate that one mechanism by which CB2 receptor activation in osteoblasts promotes breast cancer-induced osteoclastogenesis is by increasing the RANKL/OPG ratio.

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 the CB2-selective agonists JWH133 and HU308 (0.1–1 μm) after exposure to conditioned medium from human MDA-231 and MCF7 and mouse 4T1 breast cancer cells (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 photomicrographs of multinucleated TRAcP-positive osteoclasts from the experiment described in *panel B. D–F*, expression of RANKL (D) and OPG (E) as the percentage of GAPDH and RANKL/OPG ratio (F) in mouse calvarial osteoblasts in the presence or absence of the CB2-selective agonists JWH133 and HU308 (1 μm) after exposure to conditioned medium from human MDA-231 breast cancer cells (20% v/v). G, cell number in mouse calvarial osteoblasts in the presence or absence of the CB2-selective agonists JWH133 and HU308 (0.1–1 μm) 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.

CB2 Receptor Agonists Enhance Breast Cancer-induced Osteoclast Formation and Bone Resorption

To examine the direct effects of CB2 agonists on osteoclasts and their M-CSF- generated precursors, we went on to test the effects of JWH133 and HU308 on osteoclast formation in M-CSF- and RANKL-stimulated bone marrow cultures in the presence or absence of conditioned medium from a variety of human and mouse breast cancer cells (Fig. 2A). Pretreatment of M-CSF generated osteoclast precursors with the CB2 receptor-selective agonists JWH133 and HU308 at concentrations between 0.1 and 1 μm 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 osteoclastogenic effects of conditioned medium from human 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 cathepsin K, were increased by JWH133 and HU308 in these cultures (Fig. 2D). Bone resorption was also significantly enhanced by JWH133 (35% ± 14 increase) and HU308 (41% ± 1.6 increase) in similar cultures using conditioned medium from human MDA-231 (Fig. 2E). Although breast cancer cell-conditioned medium and the CB2-selective agonists enhanced the osteoclastogenic effects of M-CSF and RANKL, these factors alone and in combination were unable to support osteoclast formation in the absence of RANKL and M-CSF (Fig. 2F). Furthermore, the addition of JWH133 and HU308 to MDA-231-conditioned medium enhanced M-CSF- and RANKL-induced osteoclast formation in wild type cultures, but no effect was observed in bone marrow cultures from mice lacking CB2 receptors (CB2^−/− mice) (see Fig. 4, A and B). In accordance with this, pretreatment with the CB2-selective antagonist/inverse agonist AM630 or the G_i/o PTX for 1 h before adding HU308 and JWH133 in wild type cultures significantly inhibited osteoclast formation (see Fig. 4A). Collectively, these results suggest that JWH133 and HU308 enhance breast cancer-induced osteoclast formation by CB2 receptors in a PTX-sensitive manner.

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 to conditioned 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 agonists JWH133 and HU308 (0.1–1 μm). The average numbers of TRAcP-positive multinucleated osteoclasts with three or more nuclei in vehicle-treated control cultures are as follows: BMC, 25 ± 2; BMC/MDA-231, 45 ± 9; BMC/MCF7, 49 ± 7; BMC/4T1, 61 ± 3. C, representative photomicrographs of multinucleated TRAcP-positive osteoclasts from the experiment described in *panel B. D*, expression of the osteoclast-specific genes TRAcP, calcitonin receptor, and cathepsin K 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 breast cancer cells (20% v/v) in the presence or absence of the CB2 receptor-selective agonists JWH133 and HU308 (1 μm). E, resorbed area bone marrow cultures stimulated 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 the presence or absence of the CB2-selective agonists JWH133 and HU308 (1 μm). F, number of mature osteoclasts after exposure to conditioned medium from human MDA-231 breast cancer cells (10% v/v) in the presence or absence of the CB2-selective agonists JWH133 and HU308 (0.1–1 μm) after withdrawal of RANKL (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 breast cancer cells. CM, conditioned medium.

FIGURE 4. — **JWH133 and HU308 enhance breast cancer-induced osteoclast formation via CB2 receptors in a PTX sensitive manner and a PI3K/AKT mechanism.** A, number of osteoclasts in bone marrow cultures stimulated with mouse M-CSF and RANKL from wild type and CB2-deficient mice (*CB2*^−/−) after treatment with the CB2-selective agonists JWH133 and HU308 (1 μm) 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 μm) or the G_i/o inhibitor PTX (10 μm). 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 multinucleated TRAcP-positive osteoclasts from the experiment described in *panel A. C*, Western blot analysis of AKT phosphorylation in bone marrow cultures stimulated with 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 JWH133 and HU308 (1 μm) in the presence or absence of the CB2-selective antagonist/inverse agonist AM630 (1 μm), PI3K/AKT inhibitor LY294002 (10 μm), or PTX (10 μm). D, quantification of phosphorylated AKT at serine 473 as percentage of total AKT from the experiment described in *panel C*. *, p < 0.05 from vehicle control; +, p < 0.05 from WT vehicle in the presence of conditioned medium from MDA-231 breast cancer cells; $, p < 0.05 from AM630-, LY294002-, or PTH-treated. CM, conditioned medium; M, marker.

Role of the Akt Pathway in CB2-induced Osteoclastogenesis

The PI3/AKT signaling transduction pathway is known to play a role in osteoclastogenesis downstream of various receptors including RANK and M-CSF in osteoclasts and the parathyroid hormone (PTH) receptor in osteoblasts (39). To determine whether CB2 agonists promote AKT phosphorylation in the bone marrow microenvironment, we tested the effects of breast cancer-conditioned medium and CB2 agonists on phosphatidylinositol 3-kinase/AKT (PI3K/AKT) phosphorylation. First, we show that treatment with the CB2-selective agonists JWH133 or HU308 significantly induced the phosphorylation of AKT at serine 473, but not threonine 308, in M-CSF-stimulated bone marrow cells within 15 min (Fig. 3, A and B). Moreover, both CB2 receptor-selective agonists enhanced RANKL-induced AKT phosphorylation at serine 473 (Fig. 3, E and F). Of note, we have not detected any changes in the levels of phosphorylated or total AKT in these cultures for up to 24 h post treatment (Fig. 3, C and D). The addition of conditioned medium from MDA-231 cells enhanced AKT phosphorylation in RANKL and M-CSF-stimulated bone marrow cells, and this was further increased by JWH133 and HU308 (Fig. 4, C and D). This stimulatory effect was abolished by preincubation with the PI3K/AKT inhibitor LY294002, the G_i/o inhibitor PTX, and the CB2-selective antagonist/inverse agonist AM630 (Fig. 4, C and D). Previously, we reported that PTH, a potent stimulator of osteoblast differentiation and AKT activity (42, 43), enhances alkaline phosphatase activity in wild type calvarial osteoblasts but not in cultures from mice deficient in CB2 receptors (CB2^−/− mice) (31). Here, we examined the effects of PTH on osteoblast cultures exposed to conditioned medium from MDA-231 breast cancer cells. This showed that PTH stimulated alkaline phosphatase in osteoblast cultures and that the CB2 agonists JHW133 and HU308 further increased alkaline phosphatase levels (Fig. 5A). Similarly, PTH stimulated osteoclast formation in mouse osteoblasts/bone marrow cell cultures in the presence of MDA-231 cell-conditioned medium (Fig. 5, B and C). Osteoclast formation was further increased by the combination of PTH and the CB2 agonists JWH133 and HU308 (Fig. 5, B and C). In cultures of calvarial osteoblasts, however, JWH133 and HU308 enhanced PTH-induced phosphorylation of AKT at threonine 308 (Fig. 5, D and E). These data together demonstrate that breast cancer cell conditioned medium stimulates AKT phosphorylation in bone marrow cells and that this is increased by PTH and further increased by CB2 agonists. This illustrates that another mechanism by which the CB2 pathway enhances osteoclastogenesis is through AKT phosphorylation.

FIGURE 3. — **JWH133 and HU308 induce PI3K/AKT activation in osteoclasts in the presence and absence of RANKL.** A, Western blot analysis of AKT phosphorylation in M-CSF-generated bone marrow cells exposed to the CB2-selective agonists JWH133 or HU308 (1 μm) for 15 min. B, quantification of AKT phosphorylation from the experiment described in *panel A. C*, Western blot analysis of AKT phosphorylation in M-CSF-generated bone marrow cells exposed to the CB2-selective agonists JWH133 or HU308 (1 μm) for 24 h. D, quantification of AKT phosphorylation from the experiment described in *panel C. E*, Western blot analysis of AKT phosphorylation in bone marrow cultures stimulated with M-CSF after exposure to RANKL in the presence or absence of the CB2-selective agonists JWH133 and HU308 (1 μm). 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.

Involvement of the CB2 Receptor in Focal Osteolysis Is Mediated by Breast Cancer Cells

Next, we investigated the role of CB2 receptor activation on breast cancer-induced osteolysis in an organ culture model by using MDA-231 human breast cancer cells/mouse calvarial organ culture (Fig. 6A). The addition of MDA-231 cells to the organ cultures from wild type mice caused a dramatic decrease in calvarial bone volume, reaching 73% ± 2.9 bone loss (p < 0.01) over a 7-day-culture period (Fig. 6B). Treatment with the CB2 receptor-selective agonists JWH133 and HU308 increased osteolysis even further to 87% ± 2.7 (p < 0.05) and 92% ± 3.1 (p < 0.05), respectively (Fig. 6, B and C). However, osteolysis was reduced after treatment with the CB2-elective antagonist/inverse agonist AM630 and in organ co-cultures from CB2^−/− mice (Fig. 6B). MDA-231 cells from these organ co-cultures were not affected by JWH133 or HU308 treatment either in terms or viability (Fig. 6D) or survival, indicated by the lack of caspase-3 activation (Fig. 6E). These results demonstrate that the CB2 receptor in bone cells plays a key role in regulating local osteolysis mediated by breast cancer cells.

FIGURE 6. — **JWH133 and HU308 exacerbate breast cancer-induced osteolysis via CB2 receptor activation.** A, graphic representation of human breast cancer 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 μm) or the CB2-selective antagonist/inverse agonist AM630 (1 μm). C, representative photomicrographs of microcomputed tomography scans and TRAcP- and aniline-stained histological sections of mouse calvarial bone from the experiment described in *panel B*, showing osteolysis. D, cell number of human MDA-231 cells in breast cancer cell/mouse calvaria organ co-culture system treated with the CB2-selective agonists JWH133 and HU308 (1 μm) for 7 days. E, Western blot analysis 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 the CB2-selective agonists JWH133 and HU308 (1 μm) for 7 days. +, p < 0.05 from vehicle in the presence of MDA-231 breast cancer cells; $, p < 0.05 from WT cultures treated with JWH133 and HU308 alone in the presence of MDA-231 breast cancer cells.

Discussion

There has been an increasing interest in the potential use of cannabinoid ligands for cancer treatment. The present studies illustrate that the CB2 pathway plays a role in enhancing osteoclast activation mediated by tumor cell conditioned medium as well as exerting an inhibitory effect on growth of breast cancer cell lines. We also provide evidence for the first time showing that the stimulatory effects of CB2 are mediated not only by the effects on tumor cells but also by CB2 receptors expressed by bone cells. Evidence for this comes from the observation that CB2 agonists had no stimulatory effects on osteoclast formation in bone cell cultures from CB2-deficient mice and that the stimulatory effects of breast cancer cells on local osteolysis were attenuated in calvarial explant cultures from CB2-deficient mice.

We and others have previously shown that activation of cannabinoid receptors with agonists stimulate osteoclast formation, whereas treatment with cannabinoid receptor antagonists/inverse agonists inhibit osteoclastogenesis in vitro and ovariectomy-induced bone loss in vivo (25, 30, 40). Ofek et al. (29), on the other hand, proposed that the CB2-seletive agonist HU308 inhibits osteoclast formation in vitro and in vivo. In the present study not only have we confirmed that CB2 receptor activation with the non-psychoactive agonists JWH133 and HU308 significantly stimulates osteoclastogenesis in bone marrow cultures, but we also demonstrated that in the presence of conditioned medium from different breast cancer cell lines as well as breast cancer cells per se, these CB2-selective agonists significantly enhance osteoclast formation and osteoclastic resorption. These stimulatory effects were abolished when bone marrow cultures were prepared from mice deficient in CB2 receptors. This was mirrored in a pharmacologic inactivation context, when cultures were treated with the CB2-selective antagonist/inverse agonist AM630, pointing to the fact that any form of inactivation of the CB2 receptors suppresses osteoclastogenesis in a metastatic bone environment.

Previously, we showed that alkaline phosphatase activity, a marker of osteoblast differentiation, is enhanced by PTH in wild type calvarial osteoblasts but not in cultures from CB2^−/− mice (31). Here, PTH-induced alkaline phosphatase activity in the presence of conditioned media was further enhanced after treatment with JWH133 and HU308, supporting the fact that activation of CB2 receptors increases osteoblast differentiation in a metastatic setting. Moreover, CB2 activation stimulates osteoblast support for osteoclastogenesis by enhancing the RANKL/OPG ratio in the presence of conditioned media from breast cancer cells. It is well established that breast cancer and bone cells secrete various factors that increase RANKL and inhibit OPG expression by osteoblasts (41). Although the signaling mechanisms by which cannabinoid receptor ligands affect bone cell differentiation and function are thus far relatively unknown, the current study suggests that in M-CSF-generated osteoclast precursors and osteoblasts, cannabinoid receptor agonists enhance phosphorylation of AKT, suggesting that this may be one mechanism by which CB2 activation contributes to osteoclastic and osteoblastic effects associated with osteolysis. It is worthwhile to mention that neither HU308 nor JWH133 had any significant effects on the activation and phosphorylation of a number of key signaling proteins and transcription factors downstream of RANK receptor, namely IKKα and -β, IκB, p38, ERK1/2, MEK1/2, JNK1/2, p65NFκB, NFATc1, and cFOS (data not shown). Therefore, future studies are still needed to evaluate the mechanism by which CB2 receptor signaling in bone cells influences different aspects of AKT activity such as posttranslational modifications.

Previous investigators showed that pharmacological targeting of CB2 receptors with non-psychoactive CB2-selective agonists reduced cancer-induced bone loss and bone fracture (22, 24). However, these effects were attributed on the anti-proliferative actions of the CB2-selective agonists on cancer cells rather than on an inhibitory effect directed on osteoclasts specifically. Although we cannot exclude the possibility that the CB2 agonists studied here have inhibitory effects on tumor growth, the concentrations of these agents required to inhibit growth of tumor cells were three times higher than those that were shown here to enhance osteolysis that resulted from tumor conditioned medium. In addition, the experiments in cultured calvarial explants and cultures from CB2-deficient mice demonstrate clearly that at least part of the effect was mediated by the cannabinoid pathway in bone cells rather than the tumor. It should also be noted that the CB2-selective agents JWH133 and HU308 appeared to exacerbate osteolysis in calvarial organ co-cultures from CB2-deficient mice. Although we cannot exclude the possibility that CB2 independent effects exerted by the HU308 and JWH133 may have contributed to the effects that we observed, the present study shows that CB2 exerts bone cell-autonomous effects on differentiation of osteoclasts and exerts direct effects on osteolysis. Further studies using cultured calvarial explants and cultures from cell-specific or neuron-specific inactivation of CB2 will be required to address the relative importance of signaling by CB1 and/or other related receptors to the regulation of osteolysis described in this study.

In conclusion, our present findings suggest that inhibition of CB2 receptor signaling in the bone microenvironment may have a potential role in protecting the skeleton from the osteolysis associated with breast cancer. When combined with previous studies, these findings suggest that the skeletal CB2 receptor exhibits differential responses to treatment with CB2 ligands and raises the possibility that both CB2-selective activation and antagonism have potential efficacy in cancer-associated bone disease. The potential use of CB2 receptor agonists in cases of breast cancer with bone metastases needs to be carefully explored so that any treatment regime would take into consideration and exploit both their cell-autonomous effects in the 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, and interpreted the data, J. G. L. acquired the data, P. M. is responsible for the conception and donation of material, S. H. R. revised the article and interpreted the data, and A. I. I. made substantial contributions to conception and design, acquisition, analysis, and interpretation of data and writing and revising the article.

This work was supported in part by an European Calcified Tissue International Society/AMGEN (ECTS/AMGEN) fellowship grant (to A. I.) and a grant from the Arthritis Research UK (17713). Dr. A. I. Idris and Prof. S. H. Ralston are co-inventors on a patent claiming the use of cannabinoid receptor ligands as treatments for bone disease. Patrick Mollat is an employee of Galapagos SASU.

The abbreviations used are:

CB1: cannabinoid type 1 receptor
CB2: cannabinoid type 2 receptor
RANKL: receptor activator of NFκB ligand
M-CSF: macrophage colony stimulating factor
PTX: pertussis toxin
MDA-231: MDA-MB-231
PTH: parathyroid hormone
TRAcP: tartrate resistant acid phosphatase
α-MEM: minimum Eagle's medium
OPG: Osteoprotegerin.

References

1. Pertwee R. G., Ross R. A. (2002) Cannabinoid receptors and their ligands. Prostaglandins Leukot. Essent. Fatty Acids 66, 101–121 [DOI] [PubMed] [Google Scholar]
2. Lutz B. (2002) Molecular biology of cannabinoid receptors. Prostaglandins Leukot. Essent. Fatty Acids 66, 123–142 [DOI] [PubMed] [Google Scholar]
3. Rhee M. H., Bayewitch M., Avidor-Reiss T., Levy R., Vogel Z. (1998) Cannabinoid receptor activation differentially regulates the various adenylyl cyclase isozymes. J. Neurochem. 71, 1525–1534 [DOI] [PubMed] [Google Scholar]
4. Bouaboula M., Poinot-Chazel C., Bourrié B., Canat X., Calandra B., Rinaldi-Carmona M., Le Fur G., Casellas P. (1995) Activation of mitogen-activated protein kinases by stimulation of the central cannabinoid receptor CB1. Biochem. J. 312, 637–641 [DOI] [PMC free article] [PubMed] [Google Scholar]
5. Bouaboula M., Poinot-Chazel C., Marchand J., Canat X., Bourrié B., Rinaldi-Carmona M., Calandra B., Le Fur G., Casellas P. (1996) Signaling pathway associated with stimulation of CB2 peripheral cannabinoid receptor: involvement of both mitogen-activated protein kinase and induction of Krox-24 expression. Eur. J. Biochem. 237, 704–711 [DOI] [PubMed] [Google Scholar]
6. Sánchez M. G., Ruiz-Llorente L., Sánchez A. M., Díaz-Laviada I. (2003) Activation of phosphoinositide 3-kinase/PKB pathway by CB1 and CB2 cannabinoid receptors expressed in prostate PC-3 cells: involvement in Raf-1 stimulation and NGF induction. Cell Signal. 15, 851–859 [DOI] [PubMed] [Google Scholar]
7. Munson A. E., Harris L. S., Friedman M. A., Dewey W. L., Carchman R. A. (1975) Antineoplastic activity of cannabinoids. J. Natl. Cancer Inst. 55, 597–602 [DOI] [PubMed] [Google Scholar]
8. Caffarel M. M., Moreno-Bueno G., Cerutti C., Palacios J., Guzman M., Mechta-Grigoriou F., Sanchez C. (2008) JunD is involved in the antiproliferative effect of Delta9-tetrahydrocannabinol on human breast cancer cells. Oncogene 27, 5033–5044 [DOI] [PubMed] [Google Scholar]
9. Grimaldi C., Pisanti S., Laezza C., Malfitano A. M., Santoro A., Vitale M., Caruso M. G., Notarnicola M., Iacuzzo I., Portella G., Di Marzo V., Bifulco M. (2006) Anandamide inhibits adhesion and migration of breast cancer cells. Exp. Cell Res. 312, 363–373 [DOI] [PubMed] [Google Scholar]
10. Xian X. S., Park H., Cho Y. K., Lee I. S., Kim S. W., Choi M. G., Chung I. S., Han K. H., Park J. M. (2010) Effect of a synthetic cannabinoid agonist on the proliferation and invasion of gastric cancer cells. J. Cell. Biochem. 110, 321–332 [DOI] [PubMed] [Google Scholar]
11. Hart S., Fischer O. M., Ullrich A. (2004) Cannabinoids induce cancer cell proliferation via tumor necrosis factor α-converting enzyme (TACE/ADAM17)-mediated transactivation of the epidermal growth factor receptor. Cancer Res. 64, 1943–1950 [DOI] [PubMed] [Google Scholar]
12. McKallip R. J., Nagarkatti M., Nagarkatti P. S. (2005) Δ⁹-tetrahydrocannabinol enhances breast cancer growth and metastasis by suppression of the antitumor immune response. J. Immunol. 174, 3281–3289 [DOI] [PubMed] [Google Scholar]
13. Guzmán M. (2003) Cannabinoids: potential anticancer agents. Nat. Rev. Cancer 3, 745–755 [DOI] [PubMed] [Google Scholar]
14. Ramer R., Merkord J., Rohde H., Hinz B. (2010) Cannabidiol inhibits cancer cell invasion via up-regulation of tissue inhibitor of matrix metalloproteinases-1. Biochem. Pharmacol. 79, 955–966 [DOI] [PubMed] [Google Scholar]
15. Zhu L. X., Sharma S., Stolina M., Gardner B., Roth M. D., Tashkin D. P., Dubinett S. M. (2000) Δ⁹-Tetrahydrocannabinol inhibits antitumor immunity by a CB2 receptor-mediated, cytokine-dependent pathway. J. Immunol. 165, 373–380 [DOI] [PubMed] [Google Scholar]
16. Caffarel M. M., Andradas C., Pérez-Gómez E., Guzmán M., Sánchez C. (2012) Cannabinoids: a new hope for breast cancer therapy? Cancer Treat. Rev. 38, 911–918 [DOI] [PubMed] [Google Scholar]
17. Bifulco M., Malfitano A. M., Pisanti S., Laezza C. (2008) Endocannabinoids in endocrine and related tumours. Endocr. Relat. Cancer 15, 391–408 [DOI] [PubMed] [Google Scholar]
18. Sarfaraz S., Adhami V. M., Syed D. N., Afaq F., Mukhtar H. (2008) Cannabinoids for cancer treatment: progress and promise. Cancer Res. 68, 339–342 [DOI] [PubMed] [Google Scholar]
19. Dhopeshwarkar A., Mackie K. (2014) CB2 Cannabinoid receptors as a therapeutic target-what does the future hold? Mol. Pharmacol. 86, 430–437 [DOI] [PMC free article] [PubMed] [Google Scholar]
20. Manera C., Saccomanni G., Malfitano A. M., Bertini S., Castelli F., Laezza C., Ligresti A., Lucchesi V., Tuccinardi T., Rizzolio F., Bifulco M., Di Marzo V., Giordano A., Macchia M., Martinelli A. (2012) Rational design, synthesis. and anti-proliferative properties of new CB2-selective cannabinoid receptor ligands: an investigation of the 1,8-naphthyridin-2(1H)-one scaffold. Eur. J. Med. Chem. 52, 284–294 [DOI] [PubMed] [Google Scholar]
21. Olea-Herrero N., Vara D., Malagarie-Cazenave S., Díaz-Laviada I. (2009) Inhibition of human tumour prostate PC-3 cell growth by cannabinoids R(+)-methanandamide and JWH-015: involvement of CB2. Br. J. Cancer 101, 940–950 [DOI] [PMC free article] [PubMed] [Google Scholar]
22. Lozano-Ondoua A. N., Wright C., Vardanyan A., King T., Largent-Milnes T. M., Nelson M., Jimenez-Andrade J. M., Mantyh P. W., Vanderah T. W. (2010) A cannabinoid 2 receptor agonist attenuates bone cancer-induced pain and bone loss. Life Sci. 86, 646–653 [DOI] [PMC free article] [PubMed] [Google Scholar]
23. Curto-Reyes V., Llames S., Hidalgo A., Menéndez L., Baamonde A. (2010) Spinal and peripheral analgesic effects of the CB2 cannabinoid receptor agonist AM1241 in two models of bone cancer-induced pain. Br. J. Pharmacol. 160, 561–573 [DOI] [PMC free article] [PubMed] [Google Scholar]
24. Lozano-Ondoua A. N., Hanlon K. E., Symons-Liguori A. M., Largent-Milnes T. M., Havelin J. J., Ferland H. L. 3rd, Chandramouli A., Owusu-Ankomah M., Nikolich-Zugich T., Bloom A. P., Jimenez-Andrade J. M., King T., Porreca F., Nelson M. A., Mantyh P. W., Vanderah T. W. (2013) Disease modification of breast cancer-induced bone remodeling by cannabinoid 2 receptor agonists. J. Bone Miner. Res. 28, 92–107 [DOI] [PMC free article] [PubMed] [Google Scholar]
25. Idris A. I., van't Hof R. J., Greig I. R., Ridge S. A., Baker D., Ross R. A., Ralston S. H. (2005) Regulation of bone mass, bone loss and osteoclast activity by cannabinoid receptors. Nat. Med. 11, 774–779 [DOI] [PMC free article] [PubMed] [Google Scholar]
26. Idris A. I., Sophocleous A., Landao-Bassonga E., Canals M., Milligan G., Baker D., van't Hof R. J., Ralston S. H. (2009) Cannabinoid receptor type 1 protects against age-related osteoporosis by regulating osteoblast and adipocyte differentiation in marrow stromal cells. Cell Metab. 10, 139–147 [DOI] [PubMed] [Google Scholar]
27. Whyte L. S., Ryberg E., Sims N. A., Ridge S. A., Mackie K., Greasley P. J., Ross R. A., Rogers M. J. (2009) The putative cannabinoid receptor GPR55 affects osteoclast function in vitro and bone mass in vivo. Proc. Natl. Acad. Sci. U.S.A. 106, 16511–16516 [DOI] [PMC free article] [PubMed] [Google Scholar]
28. Tam J., Ofek O., Fride E., Ledent C., Gabet Y., Müller R., Zimmer A., Mackie K., Mechoulam R., Shohami E., Bab I. (2006) Involvement of neuronal cannabinoid receptor CB1 in regulation of bone mass and bone remodeling. Mol. Pharmacol. 70, 786–792 [DOI] [PubMed] [Google Scholar]
29. Ofek O., Karsak M., Leclerc N., Fogel M., Frenkel B., Wright K., Tam J., Attar-Namdar M., Kram V., Shohami E., Mechoulam R., Zimmer A., Bab I. (2006) Peripheral cannabinoid receptor, CB2, regulates bone mass. Proc. Natl. Acad. Sci. U.S.A. 103, 696–701 [DOI] [PMC free article] [PubMed] [Google Scholar]
30. Idris A. I., Sophocleous A., Landao-Bassonga E., van't Hof R. J., Ralston S. H. (2008) Regulation of bone mass, osteoclast function, and ovariectomy-induced bone loss by the type 2 cannabinoid receptor. Endocrinology 149, 5619–5626 [DOI] [PubMed] [Google Scholar]
31. Sophocleous A., Landao-Bassonga E., Van't Hof R. J., Idris A. I., Ralston S. H. (2011) The type 2 cannabinoid receptor regulates bone mass and ovariectomy-induced bone loss by affecting osteoblast differentiation and bone formation. Endocrinology 152, 2141–2149 [DOI] [PubMed] [Google Scholar]
32. Sophocleous A., Idris A. I., Ralston S. H. (2014) Genetic background modifies the effects of type 2 cannabinoid receptor deficiency on bone mass and bone turnover. Calcif. Tissue Int. 94, 259–268 [DOI] [PubMed] [Google Scholar]
33. Idris A. I., Krishnan M., Simic P., Landao-Bassonga E., Mollat P., Vukicevic S., Ralston S. H. (2010) Small molecule inhibitors of IkappaB kinase signaling inhibit osteoclast formation in vitro and prevent ovariectomy-induced bone loss in vivo. FASEB J. 24, 4545–4555 [DOI] [PubMed] [Google Scholar]
34. Aitken S. J., Landao-Bassonga E., Ralston S. H., Idris A. I. (2009) β2-Adrenoreceptor ligands regulate osteoclast differentiation in vitro by direct and indirect mechanisms. Arch. Biochem. Biophys. 482, 96–103 [DOI] [PubMed] [Google Scholar]
35. Bakker A., Klein-Nulend J. (2003) Osteoblast isolation from murine calvariae and long bones. In Bone Research Protocols (Helfrich M., Ralston S., eds) pp.183–198, Humana Press, Totowa, NJ [Google Scholar]
36. Idris A. I., Rojas J., Greig I. R., Van't Hof R. J., Ralston S. H. (2008) Aminobisphosphonates cause osteoblast apoptosis and inhibit bone nodule formation in vitro. Calcif. Tissue Int. 82, 191–201 [DOI] [PubMed] [Google Scholar]
37. Garret R. (2012) Assessing bone formation using mouse calvarial organ cultures. In Bone Research Protocols (Helfrich M., Ralston S., eds) 2nd Ed., Vol. 816, pp. 545–51, Humana Press, Totowa, NJ [Google Scholar]
38. Suda T., Takahashi N., Udagawa N., Jimi E., Gillespie M. T., Martin T. J. (1999) Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr. Rev. 20, 345–357 [DOI] [PubMed] [Google Scholar]
39. Ross F. P. (2006) M-CSF, c-Fms, and signaling in osteoclasts and their precursors. Ann. N.Y. Acad. Sci. 1068, 110–116 [DOI] [PubMed] [Google Scholar]
40. Whyte L. S., Ford L., Ridge S. A., Cameron G. A., Rogers M. J., Ross R. A. (2012) Cannabinoids and bone: endocannabinoids modulate human osteoclast function in vitro. Br. J. Pharmacol. 165, 2584–2597 [DOI] [PMC free article] [PubMed] [Google Scholar]
41. Thomas R. J., Guise T. A., Yin J. J., Elliott J., Horwood N. J., Martin T. J., Gillespie M. T. (1999) Breast cancer cells interact with osteoblasts to support osteoclast formation. Endocrinology 140, 4451–4458 [DOI] [PubMed] [Google Scholar]
42. Yamamoto T., Kambe F., Cao X., Lu X., Ishiguro N., Seo H. (2007) Parathyroid hormone activates phosphoinositide 3-kinase-Akt-Bad cascade in osteoblast-like cells. Bone 40, 354–359 [DOI] [PubMed] [Google Scholar]
43. Bhattacharjee R., Kaneda M., Nakahama K., Morita I. (2009) The steady-state expression of connexin43 is maintained by the PI3K/Akt in osteoblasts. BBRC 382, 440–444 [DOI] [PubMed] [Google Scholar]

[B1] 1. Pertwee R. G., Ross R. A. (2002) Cannabinoid receptors and their ligands. Prostaglandins Leukot. Essent. Fatty Acids 66, 101–121 [DOI] [PubMed] [Google Scholar]

[B2] 2. Lutz B. (2002) Molecular biology of cannabinoid receptors. Prostaglandins Leukot. Essent. Fatty Acids 66, 123–142 [DOI] [PubMed] [Google Scholar]

[B3] 3. Rhee M. H., Bayewitch M., Avidor-Reiss T., Levy R., Vogel Z. (1998) Cannabinoid receptor activation differentially regulates the various adenylyl cyclase isozymes. J. Neurochem. 71, 1525–1534 [DOI] [PubMed] [Google Scholar]

[B4] 4. Bouaboula M., Poinot-Chazel C., Bourrié B., Canat X., Calandra B., Rinaldi-Carmona M., Le Fur G., Casellas P. (1995) Activation of mitogen-activated protein kinases by stimulation of the central cannabinoid receptor CB1. Biochem. J. 312, 637–641 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B5] 5. Bouaboula M., Poinot-Chazel C., Marchand J., Canat X., Bourrié B., Rinaldi-Carmona M., Calandra B., Le Fur G., Casellas P. (1996) Signaling pathway associated with stimulation of CB2 peripheral cannabinoid receptor: involvement of both mitogen-activated protein kinase and induction of Krox-24 expression. Eur. J. Biochem. 237, 704–711 [DOI] [PubMed] [Google Scholar]

[B6] 6. Sánchez M. G., Ruiz-Llorente L., Sánchez A. M., Díaz-Laviada I. (2003) Activation of phosphoinositide 3-kinase/PKB pathway by CB1 and CB2 cannabinoid receptors expressed in prostate PC-3 cells: involvement in Raf-1 stimulation and NGF induction. Cell Signal. 15, 851–859 [DOI] [PubMed] [Google Scholar]

[B7] 7. Munson A. E., Harris L. S., Friedman M. A., Dewey W. L., Carchman R. A. (1975) Antineoplastic activity of cannabinoids. J. Natl. Cancer Inst. 55, 597–602 [DOI] [PubMed] [Google Scholar]

[B8] 8. Caffarel M. M., Moreno-Bueno G., Cerutti C., Palacios J., Guzman M., Mechta-Grigoriou F., Sanchez C. (2008) JunD is involved in the antiproliferative effect of Delta9-tetrahydrocannabinol on human breast cancer cells. Oncogene 27, 5033–5044 [DOI] [PubMed] [Google Scholar]

[B9] 9. Grimaldi C., Pisanti S., Laezza C., Malfitano A. M., Santoro A., Vitale M., Caruso M. G., Notarnicola M., Iacuzzo I., Portella G., Di Marzo V., Bifulco M. (2006) Anandamide inhibits adhesion and migration of breast cancer cells. Exp. Cell Res. 312, 363–373 [DOI] [PubMed] [Google Scholar]

[B10] 10. Xian X. S., Park H., Cho Y. K., Lee I. S., Kim S. W., Choi M. G., Chung I. S., Han K. H., Park J. M. (2010) Effect of a synthetic cannabinoid agonist on the proliferation and invasion of gastric cancer cells. J. Cell. Biochem. 110, 321–332 [DOI] [PubMed] [Google Scholar]

[B11] 11. Hart S., Fischer O. M., Ullrich A. (2004) Cannabinoids induce cancer cell proliferation via tumor necrosis factor α-converting enzyme (TACE/ADAM17)-mediated transactivation of the epidermal growth factor receptor. Cancer Res. 64, 1943–1950 [DOI] [PubMed] [Google Scholar]

[B12] 12. McKallip R. J., Nagarkatti M., Nagarkatti P. S. (2005) Δ⁹-tetrahydrocannabinol enhances breast cancer growth and metastasis by suppression of the antitumor immune response. J. Immunol. 174, 3281–3289 [DOI] [PubMed] [Google Scholar]

[B13] 13. Guzmán M. (2003) Cannabinoids: potential anticancer agents. Nat. Rev. Cancer 3, 745–755 [DOI] [PubMed] [Google Scholar]

[B14] 14. Ramer R., Merkord J., Rohde H., Hinz B. (2010) Cannabidiol inhibits cancer cell invasion via up-regulation of tissue inhibitor of matrix metalloproteinases-1. Biochem. Pharmacol. 79, 955–966 [DOI] [PubMed] [Google Scholar]

[B15] 15. Zhu L. X., Sharma S., Stolina M., Gardner B., Roth M. D., Tashkin D. P., Dubinett S. M. (2000) Δ⁹-Tetrahydrocannabinol inhibits antitumor immunity by a CB2 receptor-mediated, cytokine-dependent pathway. J. Immunol. 165, 373–380 [DOI] [PubMed] [Google Scholar]

[B16] 16. Caffarel M. M., Andradas C., Pérez-Gómez E., Guzmán M., Sánchez C. (2012) Cannabinoids: a new hope for breast cancer therapy? Cancer Treat. Rev. 38, 911–918 [DOI] [PubMed] [Google Scholar]

[B17] 17. Bifulco M., Malfitano A. M., Pisanti S., Laezza C. (2008) Endocannabinoids in endocrine and related tumours. Endocr. Relat. Cancer 15, 391–408 [DOI] [PubMed] [Google Scholar]

[B18] 18. Sarfaraz S., Adhami V. M., Syed D. N., Afaq F., Mukhtar H. (2008) Cannabinoids for cancer treatment: progress and promise. Cancer Res. 68, 339–342 [DOI] [PubMed] [Google Scholar]

[B19] 19. Dhopeshwarkar A., Mackie K. (2014) CB2 Cannabinoid receptors as a therapeutic target-what does the future hold? Mol. Pharmacol. 86, 430–437 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B20] 20. Manera C., Saccomanni G., Malfitano A. M., Bertini S., Castelli F., Laezza C., Ligresti A., Lucchesi V., Tuccinardi T., Rizzolio F., Bifulco M., Di Marzo V., Giordano A., Macchia M., Martinelli A. (2012) Rational design, synthesis. and anti-proliferative properties of new CB2-selective cannabinoid receptor ligands: an investigation of the 1,8-naphthyridin-2(1H)-one scaffold. Eur. J. Med. Chem. 52, 284–294 [DOI] [PubMed] [Google Scholar]

[B21] 21. Olea-Herrero N., Vara D., Malagarie-Cazenave S., Díaz-Laviada I. (2009) Inhibition of human tumour prostate PC-3 cell growth by cannabinoids R(+)-methanandamide and JWH-015: involvement of CB2. Br. J. Cancer 101, 940–950 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B22] 22. Lozano-Ondoua A. N., Wright C., Vardanyan A., King T., Largent-Milnes T. M., Nelson M., Jimenez-Andrade J. M., Mantyh P. W., Vanderah T. W. (2010) A cannabinoid 2 receptor agonist attenuates bone cancer-induced pain and bone loss. Life Sci. 86, 646–653 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B23] 23. Curto-Reyes V., Llames S., Hidalgo A., Menéndez L., Baamonde A. (2010) Spinal and peripheral analgesic effects of the CB2 cannabinoid receptor agonist AM1241 in two models of bone cancer-induced pain. Br. J. Pharmacol. 160, 561–573 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B24] 24. Lozano-Ondoua A. N., Hanlon K. E., Symons-Liguori A. M., Largent-Milnes T. M., Havelin J. J., Ferland H. L. 3rd, Chandramouli A., Owusu-Ankomah M., Nikolich-Zugich T., Bloom A. P., Jimenez-Andrade J. M., King T., Porreca F., Nelson M. A., Mantyh P. W., Vanderah T. W. (2013) Disease modification of breast cancer-induced bone remodeling by cannabinoid 2 receptor agonists. J. Bone Miner. Res. 28, 92–107 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B25] 25. Idris A. I., van't Hof R. J., Greig I. R., Ridge S. A., Baker D., Ross R. A., Ralston S. H. (2005) Regulation of bone mass, bone loss and osteoclast activity by cannabinoid receptors. Nat. Med. 11, 774–779 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B26] 26. Idris A. I., Sophocleous A., Landao-Bassonga E., Canals M., Milligan G., Baker D., van't Hof R. J., Ralston S. H. (2009) Cannabinoid receptor type 1 protects against age-related osteoporosis by regulating osteoblast and adipocyte differentiation in marrow stromal cells. Cell Metab. 10, 139–147 [DOI] [PubMed] [Google Scholar]

[B27] 27. Whyte L. S., Ryberg E., Sims N. A., Ridge S. A., Mackie K., Greasley P. J., Ross R. A., Rogers M. J. (2009) The putative cannabinoid receptor GPR55 affects osteoclast function in vitro and bone mass in vivo. Proc. Natl. Acad. Sci. U.S.A. 106, 16511–16516 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B28] 28. Tam J., Ofek O., Fride E., Ledent C., Gabet Y., Müller R., Zimmer A., Mackie K., Mechoulam R., Shohami E., Bab I. (2006) Involvement of neuronal cannabinoid receptor CB1 in regulation of bone mass and bone remodeling. Mol. Pharmacol. 70, 786–792 [DOI] [PubMed] [Google Scholar]

[B29] 29. Ofek O., Karsak M., Leclerc N., Fogel M., Frenkel B., Wright K., Tam J., Attar-Namdar M., Kram V., Shohami E., Mechoulam R., Zimmer A., Bab I. (2006) Peripheral cannabinoid receptor, CB2, regulates bone mass. Proc. Natl. Acad. Sci. U.S.A. 103, 696–701 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B30] 30. Idris A. I., Sophocleous A., Landao-Bassonga E., van't Hof R. J., Ralston S. H. (2008) Regulation of bone mass, osteoclast function, and ovariectomy-induced bone loss by the type 2 cannabinoid receptor. Endocrinology 149, 5619–5626 [DOI] [PubMed] [Google Scholar]

[B31] 31. Sophocleous A., Landao-Bassonga E., Van't Hof R. J., Idris A. I., Ralston S. H. (2011) The type 2 cannabinoid receptor regulates bone mass and ovariectomy-induced bone loss by affecting osteoblast differentiation and bone formation. Endocrinology 152, 2141–2149 [DOI] [PubMed] [Google Scholar]

[B32] 32. Sophocleous A., Idris A. I., Ralston S. H. (2014) Genetic background modifies the effects of type 2 cannabinoid receptor deficiency on bone mass and bone turnover. Calcif. Tissue Int. 94, 259–268 [DOI] [PubMed] [Google Scholar]

[B33] 33. Idris A. I., Krishnan M., Simic P., Landao-Bassonga E., Mollat P., Vukicevic S., Ralston S. H. (2010) Small molecule inhibitors of IkappaB kinase signaling inhibit osteoclast formation in vitro and prevent ovariectomy-induced bone loss in vivo. FASEB J. 24, 4545–4555 [DOI] [PubMed] [Google Scholar]

[B34] 34. Aitken S. J., Landao-Bassonga E., Ralston S. H., Idris A. I. (2009) β2-Adrenoreceptor ligands regulate osteoclast differentiation in vitro by direct and indirect mechanisms. Arch. Biochem. Biophys. 482, 96–103 [DOI] [PubMed] [Google Scholar]

[B35] 35. Bakker A., Klein-Nulend J. (2003) Osteoblast isolation from murine calvariae and long bones. In Bone Research Protocols (Helfrich M., Ralston S., eds) pp.183–198, Humana Press, Totowa, NJ [Google Scholar]

[B36] 36. Idris A. I., Rojas J., Greig I. R., Van't Hof R. J., Ralston S. H. (2008) Aminobisphosphonates cause osteoblast apoptosis and inhibit bone nodule formation in vitro. Calcif. Tissue Int. 82, 191–201 [DOI] [PubMed] [Google Scholar]

[B37] 37. Garret R. (2012) Assessing bone formation using mouse calvarial organ cultures. In Bone Research Protocols (Helfrich M., Ralston S., eds) 2nd Ed., Vol. 816, pp. 545–51, Humana Press, Totowa, NJ [Google Scholar]

[B38] 38. Suda T., Takahashi N., Udagawa N., Jimi E., Gillespie M. T., Martin T. J. (1999) Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr. Rev. 20, 345–357 [DOI] [PubMed] [Google Scholar]

[B39] 39. Ross F. P. (2006) M-CSF, c-Fms, and signaling in osteoclasts and their precursors. Ann. N.Y. Acad. Sci. 1068, 110–116 [DOI] [PubMed] [Google Scholar]

[B40] 40. Whyte L. S., Ford L., Ridge S. A., Cameron G. A., Rogers M. J., Ross R. A. (2012) Cannabinoids and bone: endocannabinoids modulate human osteoclast function in vitro. Br. J. Pharmacol. 165, 2584–2597 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B41] 41. Thomas R. J., Guise T. A., Yin J. J., Elliott J., Horwood N. J., Martin T. J., Gillespie M. T. (1999) Breast cancer cells interact with osteoblasts to support osteoclast formation. Endocrinology 140, 4451–4458 [DOI] [PubMed] [Google Scholar]

[B42] 42. Yamamoto T., Kambe F., Cao X., Lu X., Ishiguro N., Seo H. (2007) Parathyroid hormone activates phosphoinositide 3-kinase-Akt-Bad cascade in osteoblast-like cells. Bone 40, 354–359 [DOI] [PubMed] [Google Scholar]

[B43] 43. Bhattacharjee R., Kaneda M., Nakahama K., Morita I. (2009) The steady-state expression of connexin43 is maintained by the PI3K/Akt in osteoblasts. BBRC 382, 440–444 [DOI] [PubMed] [Google Scholar]

PERMALINK

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

Antonia Sophocleous

Silvia Marino

John G Logan

Patrick Mollat

Stuart H Ralston

Aymen I Idris