Non–small cell lung cancer (NSCLC) is the leading cause of cancer deaths worldwide; however, only limited therapeutic treatments are available. Hence, we investigated the role of cannabinoid receptors, CB1 and CB2, as novel therapeutic targets against NSCLC. We observed expression of CB1 (24%) and CB2 (55%) in NSCLC patients. Furthermore, we have shown that the treatment of NSCLC cell lines (A549 and SW-1573) with CB1/CB2- and CB2-specific agonists Win55,212-2 and JWH-015, respectively, significantly attenuated random as well as growth factor-directed in vitro chemotaxis and chemoinvasion in these cells. We also observed significant reduction in focal adhesion complex, which plays an important role in migration, upon treatment with both JWH-015 and Win55,212-2. In addition, pretreatment with CB1/CB2 selective antagonists, AM251 and AM630, prior to JWH-015 and Win55,212-2 treatments, attenuated the agonist-mediated inhibition of in vitro chemotaxis and chemoinvasion. In addition, both CB1 and CB2 agonists Win55,212-2 and JWH-133, respectively, significantly inhibited in vivo tumor growth and lung metastasis (∼50%). These effects were receptor mediated, as pretreatment with CB1/CB2 antagonists abrogated CB1/CB2 agonist–mediated effects on tumor growth and metastasis. Reduced proliferation and vascularization, along with increased apoptosis, were observed in tumors obtained from animals treated with JWH-133 and Win55,212-2. Upon further elucidation into the molecular mechanism, we observed that both CB1 and CB2 agonists inhibited phosphorylation of AKT, a key signaling molecule controlling cell survival, migration, and apoptosis, and reduced matrix metalloproteinase 9 expression and activity. These results suggest that CB1 and CB2 could be used as novel therapeutic targets against NSCLC. Cancer Prev Res; 4(1); 65–75. ©2010 AACR.

Non–small cell lung cancer (NSCLC), particularly metastatic lung cancer that accounts for approximately 85% of lung cancer cases, is the leading cause of cancer-related mortality in the United States (1). Less than 15% of patients survive beyond 5 years after diagnosis. Overexpression of epidermal growth factor receptor (EGFR) is associated with a majority of NSCLC and has been implicated in the process of malignant transformation by promoting cell proliferation, cell survival, and motility (2, 3). A series of targets and therapeutic strategies for the treatment of lung cancer is currently being investigated. All patients ultimately develop resistance against these agents, including chemotherapy, possibly due to abnormal signal transduction and high EGFR expression levels (4, 5). Hence, abrogation of EGFR action is considered a promising strategy for anticancer therapy (6). However, recent experimental evidence suggests that cancer cells may escape from growth inhibition by alternative growth pathways or by constitutive activation of downstream signaling effectors in the presence of direct EGFR inhibitors (7). Consequently, there is a need for alternate therapy in which other receptors specifically expressed on tumor cells can be targeted to abrogate EGFR-mediated signaling events directly or indirectly. In the present study, therefore, we analyzed the role of cannabinoid receptors CB1 and CB2 in NSCLC growth and metastasis.

There are 3 general types of cannabinoids: phytocannabinoids, Δ9-tetrahydrocannabinol (THC), and carbinodiol, are derived from plants; endogenous cannabinoids, 2AG and AEA, which are produced inside the body; and synthetic cannabinoids, JWH-133/JWH-015, CP-55, and Win55,212-2. These cannabinoids bind to 2 different cell surface G-protein–coupled receptors (GPCR), CB1 and CB2. CB1 receptor is predominantly expressed in the central nervous system (8, 9), whereas CB2 receptor is expressed by immune cells (10). Recently, CB1 and CB2 have been shown to be overexpressed on tumor cells compared with normal cells in various types of cancers, such as breast (11) and liver cancers (12), and therefore could be used as novel targets for cancer. In addition, several cannabinoids, including THC and cannabidiol, and synthetic cannabinoid-agonists, such as JWH-133, Win55,212-2, were shown to inhibit tumor growth and progression of several types of cancers, including glioma, glioblastoma multiforme, breast, prostate, and colon carcinomas, leukemia, and lymphoid tumors (13, 14). In addition, synthetic cannabinoids were shown to inhibit breast tumor growth in MMTV-neu and polyoma middle T oncoprotein (PyMT) breast cancer transgenic mouse models (11, 15). However, not much is known about the role of synthetic cannabinoids in lung cancer growth and metastasis.

The use of cannabinoid-related drugs for medicinal purposes can be limited because of concerns of their psychotropic effects. However, synthetic cannabinoids have been shown to possess limited psychotropic effects. Also, cannabinoids have been shown to possess minimal adverse effects and are currently being used for medical purposes in various countries. Another important feature of synthetic cannabinoids is that they have been shown to be well tolerated by in vivo studies and do not present any generalized toxic effects, as observed in most conventional chemotherapeutic agents. They have been used for the treatment of various disease conditions, such as wasting and vomiting associated with cancer chemotherapy, AIDS, multiple sclerosis, and Parkinson's disease (16).

In the present study, we analyzed the expression of CB1 and CB2 receptors in human lung cancer patient samples over their normal counterparts. We further analyzed the activity of CB1 and CB2 synthetic agonists on NSCLC migration in vitro as well as in vivo growth and metastasis. We also determined the CB1/CB2 mechanisms that regulate tumor growth and migration of NSCLC. This study provides novel insights about the role of cannabinoid receptors CB1 and CB2 in NSCLC growth and metastasis.

Cell culture

Human NSCLC cells, A549 cells, and SW-1573 cells were obtained from American Type Culture Collection (ATCC). The cell lines have been characterized by ATCC and we have not done any further characterization. Cells were cultured and maintained in DMEM or RPMI 1640, supplemented with 10% heat-inactivated FBS, 5 units/mL penicillin, and 5 mg/mL streptomycin.

Cell viability and proliferation

Cells grown for 24 hours at a density of 1 × 104 cells per 100 μL of complete medium in 96-well plate were incubated with JWH-015/Win55,212-2 (1–20 μmol/L; Tocris Cookson) for 24 hours. Cell viability was determined by the CellTiter 96 Aqueous One Solution Reagent (MTS; Promega), according to the manufacturer's instructions.

For proliferation assay, 2 × 105 cells per well were plated in 6-well plates in the presence of different concentrations of JWH-015 or Win55,212-2 or vehicle in the presence of EGF (100 ng/mL). Cells were trypsinized and counted using a hematocytometer after 24, 48, and 72 hours (11, 17).

Cell scattering assay

A total of 1 × 104 cells per well were plated in 6-well culture plates. Once they formed small colonies, they were serum starved for 24 hours and pretreated with JWH-015 or Win55,212-2 for 30 minutes prior to stimulation with EGF (10 ng/mL) and incubated for another 48 hours. Cell scattering was examined by phase-contrast microscopy and photographed.

Cell migration and invasion assays

A series of assays were used to study the effect of cannabinoid receptor activation on migratory and invasive potential of NSCLC cells. In wound-healing assay, cells were plated at 70% confluence in 10% serum-DMEM. At 24 hours after seeding, the monolayers were wounded by scoring with a sterile plastic 200-μL micropipette tip, washed, and fed with serum-free DMEM (0.1% FBS). After culturing the cells in the presence or absence of JWH-015 and Win55,212-2 with pertussis toxin (PTX) and EGF (10 ng/mL) for 72 hours, cells were fixed with 4% paraformaldehyde in PBS for 5 minutes at room temperature and photographed using a low-magnification phase-contrast microscope. The extent of migration into the wound area was evaluated qualitatively.

For Transwell chamber (Corning-Costar) migration assays, 100 μL of 1 × 106 cells were pretreated with JWH-015 or Win55,212-2 prior to antagonist (PTX, AM630, or AM251; Tocris Cookson) treatment and were seeded in the upper chamber of Transwells in serum-free medium as described previously (10). DMEM with 10% serum or EGF (10 ng/mL) in the presence of corresponding cannabinoid agonist/antagonist was added to the lower chamber. Cells that migrated into the lower chamber were counted 12 hours after stimulation with EGF.

In in vitro invasion assay, precoated Matrigel 24-well invasion chambers (BD Biosciences) were used. Treatment of the cells was similar to migration assay as described earlier. After 24-hour stimulation with EGF, cells adherent to the outer surface of the filter membrane separating upper and lower chambers were fixed and stained with Harris modified Fisher hematoxylin (Fisher) and the cells were counted (11, 17).

Immunoblot analysis

Extraction of proteins from cultured cells and immunoblot analysis were conducted as per standard protocols. Briefly, the cells were lysed in cell lysis buffer, extracts were clarified by centrifugation at 12,000 rpm, and the supernatants were subjected to immunoblot analysis. Primary antibodies directed against phospho-AKT and AKT (Cell Signaling Technology) were used at dilutions of 1:1,000. After washing 3 times each for 10 minutes in 1× TBST, blots were exposed to the secondary antibody (anti-mouse or anti-rabbit IgG-HRP; Santa Cruz Biotechnology Inc) at a dilution of 1:2,000 and visualized using ECL chemiluminescence detection system (Amersham).

Immunofluorescence microscopy

Cells were grown overnight on tissue culture–treated chambered slides (BD Biosciences), treated with cannabinoids, and stimulated for 3 hours with EGF (10 ng/mL) in serum-free medium, washed and fixed with 4% paraformaldehyde, permeabilized in PBS containing 0.2% Triton, and blocked with 3% bovine serum albumin (BSA). Cells were incubated with Texas Red–labeled phalloidin (Molecular Probes) and FITC-labeled antivinculin (Sigma) for 2 hours at room temperature and washed. After final washes and mounting, cells were examined using a confocal microscope.

ELISA

Matrix metalloproteinase (MMP) 9 levels were measured in supernatants from cells treated with different concentrations of JWH-015 and Win55,212-2 for 24 hours in serum-free growth medium. Protein levels in the supernatants were assayed using a MMP-9 ELISA Kit (R&D Systems) following the manufacturer's instruction. Optical density was measured at 450 nm. MMP-9 concentration was calculated by comparing the data to the known standards for MMP-9 proteins.

Mouse model study for tumor xenograft growth and metastasis

Subcutaneous tumors were generated in SCID (severe combined immunodeficient mice) CB-17 mice (Charles River Laboratories Inc.) by subcutaneous injection of 3 × 106 viable A549 cells in PBS. To grow pulmonary tumor colonies, mice were given injections of 5 × 105 viable tumor cells through the lateral tail vein. Once the tumors reached an average palpable size in subcutaneous model and 7 days after cell injection in metastatic model, animals were assigned randomly to various groups and injected daily with different doses of cannabinoid agonists JWH-133 (1 mg/kg/d) and Win55,212-2 (0.1 mg/kg/d) alone and in combination with CB2 specific antagonists SR144528 (Sanofi Recherche) at a dose of 1.0 mg/kg/d and CB1/CB2 nonspecific antagonist AM281 (analogue of AM251 for use in animal studies) at a dose 0.1 mg/kg/d peritumorally or intraperitoneally for 28 days. Tumor sizes were measured with external calipers weekly in 2 dimensions throughout the study and calculated as follows: tumor volume = length × (width)2/2 (11, 17).

Immunohistochemistry

Lung carcinoma tissue microarray (TMA) slides were obtained from Imgenex. Antigen retrieval was carried out by heat-induced antigen retrieval, in which slides were placed in Dako TRS solution (pH 6.1) for 25 minutes at 94°C and cooled for 15 minutes. Slides were then placed on Dako Autostainer, an immunostaining system, for use with immunohistochemistry. Slides were incubated for 60 minutes in CB1 or CB2 antibodies (1:200) or their respective peptides and detected using a labeled polymer system, ImmPRESS Anti-Rabbit Ig (peroxidase) kit (Vector Laboratories), as per manufacturer's protocol. Staining was visualized with DAB chromogen. Slides were then counterstained in Richard Allen hematoxylin, dehydrated through graded ethanol solutions, and coverslipped. Sections were examined for CB1 and CB2 expression and immunoreactivity was scored as 0 (no or weak focal expression) and 1 (strong diffuse expression).

Samples from tumor xenografts of mice were dissected and embedded in OCT (Tissue-Tek). Standard immunohistochemistry techniques were used as per the manufacturer's recommendations (Vector Laboratories), using the primary antibody against Ki-67 (Neomarkers) at a dilution of 1/100 and CD31 (BD Pharmingen) at a dilution of 1:50 for 60 minutes at room temperature. Vectastain Elite ABC reagents (Vector Laboratories), using avidin DH:biotinylated horseradish peroxidase H complex with 3,3′-diaminobenzidine (Polysciences) and Mayer's hematoxylin (Fisher Scientific), was used for detection of the bound antibodies. The nuclei for Ki-67 staining and CD31 expression in vessels were quantitated in 5 random microscopic (×10) fields per tumor. For TUNEL assay, frozen tumor sections from treated mice were analyzed by the terminal uridine deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay using the In Situ Cell Death Detection Kit following the instructions of the manufacturer (Roche). The number of apoptotic nuclei per area was counted in 20 visual fields per section under a fluorescence microscope. The results were expressed as the percentage change, with vehicle-treated samples normalized to 100% (11).

Statistical analysis

Results were analyzed using a 2-tailed Student's t test to assess statistical significance. Values of P < 0.05 were considered statistically significant.

Cannabinoid receptors are expressed in human lung cancers

Cannabinoid receptors have been shown to be overexpressed in different cancers such as skin and breast cancers (18, 19). However, to the best of our knowledge, their expression in lung carcinomas is not documented in the literature. Therefore, we first investigated cannabinoid receptor expression in a set of non–small cell lung carcinomas and showed that CB1 expression was seen in 24% (7/29) whereas CB2 was expressed in 55% (16/29) of cases. To ascertain the specificity of the cannabinoid receptor antibodies used for detection of the 2 receptors, antigen preabsorption experiments were carried out with the corresponding blocking peptides. The peptides blocked anti-CB1 and anti-CB2 antibody binding, both in cancerous and normal tissue samples (Fig. 1).

Synthetic cannabinoids inhibit proliferation of NSCLC cell lines in vitro

EGFR overexpression and overactivation are reportedly associated with resistance of lung cancer to conventional chemotherapy. Hence, the effect of CB1/CB2 and CB2 specific cannabinoids Win55,212-2 and JWH-015, respectively, was analyzed on direct EGF-induced and serum-mediated proliferation in NSCLC cell lines A549 and SW-1573 cells. CB1 and CB2 are receptors that belong to the class of GPCRs. Reports suggest cross talks between GPCRs and EGFR, with GPCRs are shown to transactivate the EGFRs (20). In the present study, we observed a dose-dependent inhibition of proliferation of the NSCLC cell line A549 cells with both Win55,212-2 and JWH-015. The CB1/CB2 agonist Win55,212-2 significantly attenuated the proliferation of A549 in response to serum and EGF by 70% and 60%, respectively (Fig. 1). A corresponding dose of the CB2 specific agonist JWH-015 also reduced the number of viable cells significantly by 40% and 50% in a dose-dependent manner in serum and EGF-stimulated A549 cells (Fig. 2A). Analogous inhibitory tendencies were observed on the proliferation of SW-1573 cell line (data not shown).

Synthetic cannabinoids inhibit cell migration and invasion of NSCLC

The ability of tumor cells to migrate from the site of the primary tumor and to invade surrounding tissues is a prerequisite for metastasis, which is the major cause of cancer-related mortality. Hence, to evaluate the therapeutic potential of cannabinoid receptors as possible targets, the effects of CB1 and CB2 agonists on EGF-induced cell migration and invasiveness were investigated. It was observed that cannabinoid agonists pretreatment impaired the cells to generate initial protrusions and scatter in response to EGF, resulting in a rounded morphology (Fig. 2B). Furthermore, different in vitro methods such as scratch wound assay, Transwell migration, and invasion assays were employed to study the effect of cannabinoid treatment on EGF-induced migration of NSCLC cell lines. As shown in Figure 2C, Win55,212-2 and JWH-015 significantly decreased EGF-induced colonization of the wound areas. The expansion of cell population upon EGF stimulation was quantified by calculating the percentage recolonization of the scratched wound surface over 72 hours. Because CB1 and CB2 belong to the class of GPCRs (8, 9), we pretreated cells with PTX, which inhibits GPCR signaling. As shown in Figure 2C, PTX pretreatment abrogated Win55,212-2- and JWH-015–mediated inhibitory effects on EGF-induced wound healing. These results implicate the involvement of cannabinoid receptors CB1 and CB2 in the inhibition of EGF-induced wound healing.

To understand the mechanism of CB1 and CB2 receptor–mediated inhibition of migration, we analyzed the effect of cannabinoid receptor agonists on the formation of focal adhesions induced with EGF. Focal adhesions are sites of tight adhesion and provide a structural link between the actin cytoskeleton and the extracellular matrix (ECM). Focal adhesions were visualized through immunofluorescent staining for vinculin, a membrane-cytoskeletal protein in focal adhesion plaques. Control and experimental cells exhibit focal adhesions at the periphery upon stimulation with EGF (Fig. 3). However, treatment of the cells with JWH-015 and Win55,212-2 prior to stimulation with EGF caused significant reduction in the number of focal adhesions of the cells. Furthermore, the polymerization state of actin, visualized as stress fibers stained with phalloidin actin, another important component related to focal adhesion formation and cell migration, was also found to be decreased in the cannabinoid-treated cells.

To confirm the CB1 and CB2 receptor–mediated inhibitory effects on EGF-induced migration and invasion, Transwell migration studies were conducted with NSCLC cells pretreated with CB1 and CB2 specific antagonists, prior to exposure with synthetic cannabinoid agonists. We observed that synthetic cannabinoid agonists inhibited EGF-induced migration and invasion of NSCLC cells in a dose-responsive manner (Fig. 4A). Both the agonists Win55,212-2 and JWH-015 significantly inhibited EGF-induced Transwell migration as well invasion by ∼50% in nanomolar doses in comparison with the corresponding vehicle-treated cells. Furthermore, pretreatment with CB1 and CB2 specific antagonists AM281 and AM630, respectively, prior to the agonist treatment, resulted in abrogation of the cannabinoid-induced inhibitory effect on EGF-stimulated migration and invasion (Fig. 4C and D). The concentrations of cannabinoid receptor agonists and antagonists used in this study did not have any significant effect on the viability of the cell lines, as was confirmed by Trypan blue staining of the cells in upper chambers of the Transwell inserts as well as by viability (MTS) assay (data not shown). In view of previous reports in which cannabinoids have been reported to induce migration in different cell lines (21–23), we investigated the induction of chemotaxis in A549 and SW-1573 cells by JWH-015 and Win 55,212-2 and found that there was no increased migration over the spontaneous migration observed in control unstimulated cells (data not shown).

Synthetic cannabinoids inhibit NSCLC tumor growth and metastasis in vivo

Because regression in experimental animal tumor models represents an important endpoint of clinical relevance, we evaluated the ability of cannabinoids to inhibit tumor growth and lung metastasis in vivo. To analyze the antitumor effects of cannabinoids in vivo, tumors generated by subcutaneous inoculation of the highly malignant NSCLC cell line (A549) were treated with vehicle or CB2 selective agonist JWH-133 (analogue of JWH-015 used for animal studies) and CB1/CB2 agonist Win55,212-2. Furthermore, to characterize the role of cannabinoid receptors in this growth inhibition, CB1 selective antagonist AM281 (analogue of AM251 used for animal studies) and CB2 selective antagonist SR144528 were administered in different combinations along with the agonist. Cannabinoid administration blocked the subcutaneous growth of tumor cells in vivo (Fig. 5A), ∼70% with JWH-133 (1 mg/kg/d) and ∼50% with Win55,212-2 (0.1 mg/kg/d). The attenuation of tumor growth with the cannabinoids was abrogated when the cannabinoids were administered in the presence of antagonists, suggesting the direct implication of the cannabinoid receptors in this growth inhibitory effect. As the tumor growth is directly correlated to the proliferation of cells, which depends on adequate vascularization required for oxygen and nutrient supply to the dividing cells, we analyzed important cellular parameters such as proliferation, angiogenesis, and apoptosis within the tumor tissues. In tumors from animals treated with both CB1/CB2 and CB2 specific agonists, we observed a significant reduction in Ki-67 immunostaining (marker for proliferation) as well as CD31 immunostaining (marker for angiogenesis). Besides this, an increase in apoptosis was observed, as analyzed with tunnel staining shown as percentage changes with respect to the untreated control animals (Fig. 5B).

Given the inhibition of migration and invasion of NSCLC cells in vitro, we evaluated the effect of cannabinoid agonist treatment on in vivo lung metastasis in SCID mice. We observed significant surface lung metastasis resulting in increased lung weight in animals inoculated with A549 cells via lateral tail vein injection (Fig. 5C). Both CB1/CB2 agonists Win55,212-2 (∼60%) and CB2 JWH-133 (∼50%) treatment attenuated lung metastasis in experimental animals over control animals. Furthermore, coadministration of CB1 and CB2 selective antagonists along with agonists abrogated this inhibitory effect, as evident by the surface lung metastases and reversal of lung weight in comparison with vehicle-treated animal (Fig. 5C). Of interest, no significant alterations in physiologic parameters such as body weight and liver weight could be ascribed to cannabinoid receptor agonist or antagonist administration, except for nonsignificant reduction in body weight with JWH-133 (data not shown).

Synthetic cannabinoids inhibit AKT/PKB phosphorylation and secretion of MMP-9 in human NSCLC cell lines

We next sought to determine CB1/CB2-mediated signaling mechanisms that inhibit tumor growth. We observed reduction in the tyrosine phosphorylation of AKT in cells pretreated with Win55,212-2 and JWH-015 prior to EGF stimulations (Fig. 6A and B). However, cannabinoid pretreatment did not change AKT protein level. AKT is the key survival molecule that is essential for EGF-induced cell migration, invasion, and proliferation (17, 24, 25).

In our study, we also observed that both Win55,212-2 and JWH-015 significantly inhibited the secretion and activity of MMP-9 in PMA-stimulated NSCLC cell lines, A549, measured through ELISA (Fig. 6C) and zymography (data not shown). Of importance, the reduction observed in the secretion of MMP-9 was significantly inhibited when the treatment was carried out in the presence of PTX, an inhibitor for GPCRs, directly implicating the involvement of CB1 and CB2 receptors. Matrix metalloproteinase are important proteins that play a critical role in cancer growth and metastasis. Significant upregulation and coexpression of MMP-9 are observed in different cancers (26–28).

Lung cancer is the leading cause of cancer-related mortality for both men and women in United States and is estimated to remain the most fatal cancer-related malignancy (1). Little improvement in the efficacy of chemotherapy has been made in the last 20 years, usually attributed to the overexpression and overactivity of EGFR in NSCLC (4, 29–31). However, the use of selective EGFR tyrosine kinase inhibitors (gefitinib) and monoclonal antibodies against EGFR toward the treatment of lung cancer has continuously failed (32, 33). Therefore, additional alternative therapies with low toxicity and increased efficacy should be explored. Although recent studies suggest that nonpsychoactive synthetic cannabinoids possess antitumor effects against various tumors, including breast cancer, not much is known about the effects of synthetic CB1/CB2 agonists on NSCLC growth and metastasis. In the present study, we analyzed the antitumorigenic and antimetastasis effects of CB1/CB2 agonists Win55,212-2 and CB2 agonists JWH-015 or JWH-133 on NSCLC cells. We report for the first time that CB1/CB2 synthetic agonists significantly inhibited NSCLC migration and growth in vitro and in vivo. In addition, we have shown that these effects are abrogated by CB1/CB2 antagonists, indicating the direct role of CB1/CB2 receptors in regulating NSCLC growth and metastasis.

Synthetic cannabinoids exert a wide variety of biological effects by mimicking endogenous compounds (endocannabinoids). However, synthetic cannabinoids do not possess psychotropic activity compared with endocannabinoids. Furthermore, endocannabinoids are metabolically unstable, as they are degraded by endogenous enzymes such as fatty acid amide hydrolase. Synthetic cannabinoids have also been shown to possess minimal adverse effects and are currently been used for medical purposes in various countries.

Synthetic cannabinoids have been shown to exert their effects through 2 cannabinoid-specific GPCRs, CB1 and CB2. Synthetic cannabinoids, especially JWH, has been shown to specifically bind to CB2 receptor whereas endocannabinoids bind to CB1/CB2 and other GPCRs, especially transient receptor potential channels (TRPV1). In this study, we have shown by TMA for the first time overexpression of CB1 and CB2 in primary human lung tumor samples. The expression of CB1 (∼24%) and CB2 (∼55%) receptors was higher in NSCLC than in normal lung tissue. Recently, CB1 and CB2 also have been reported to be overexpressed in various cancers, such as prostate and skin cancers and hepatocellular carcinoma (12, 14, 19).

In the present investigation, we show for the first time that synthetic cannabinoids, which do not possess psychoactivity, significantly inhibit in vitro as well as in vivo growth and metastasis of NSCLC cells. Pretreatment of NSCLC cells with CB2 and CB1/CB2 agonists, JWH-015 and Win55,212-2 respectively, prior to stimulation with EGF significantly inhibited EGF-induced proliferation, migration, and invasion in NSCLC cell lines. Although conflicting data are available regarding modulation of migration with cannabinoids based on the cell type, not much is known about the role of synthetic cannabinoids in blocking EGF-induced migration (17, 34). The in vitro inhibitory effects were further confirmed in vivo in murine models of xenograft tumor growth and lung metastasis in which SCID mice were injected with human lung adenocarcinoma cell line A549. Treatment of animals with JWH-133 and Win55-212, 2 resulted in significant inhibition of tumor growth and metastases in comparison with animals treated with vehicle alone. Win55,212-2 showed similar results with doses of 0.1 and 1 mg/kg/d, whereas JWH-133 was more effective at a higher dose of 1 mg/kg/d rather than 0.1 mg/kg/d (data not shown). This may be attributed to the fact that Win55,212-2 acts through both CB1 and CB2 receptors whereas JWH-133 being a CB2 specific ligand acts only through CB2 receptors. A simultaneous delivery of these agonists along with the CB1 and CB2 specific antagonists, AM281 and SR144528, respectively, abrogated the growth and metastasis inhibitory effects observed in the synthetic agonist-only treated groups. This suggests that the growth inhibitory effects of synthetic agonists are mediated by CB1 and CB2 receptors. Direct role of CB1 and CB2 receptors in inhibiting growth of different cancers by cannabinoids has been reported (19, 35, 36).

The molecular mechanism of cannabinoid-mediated inhibition of tumor growth and metastasis is not well known. Although THC downregulates RAS-MAPK/ERK and phosphatidylinositol 3-kinase (P13K)-AKT cell survival pathways leading to the induction of apoptosis in various types of cancers (17, 37), not much is known about the role of synthetic cannabinoids in lung cancer. We observed that CB1/CB2 and CB2 synthetic agonists significantly reduced the phosphorylation of the key signaling molecule AKT, a process that is known to play an important role in migration by regulating actin-myosin contractility and the focal adhesion formation (38, 39). Reduction in phosphorylation of AKT observed with both the cannabinoids JWH-015 and Win55,212-2 might be the plausible cause for reduced migration and metastases observed in in vivo studies. Previous studies have shown that inhibition of AKT phosphorylation results in a global negative effect on cell motility (40, 41). We also observed an inhibition in focal adhesion formation in response to EGF and serum stimulation in NSCLC cells along with a decrease in stress fibers. Focal adhesions are the primary subcellular macromolecules, the closest contacts between cells and the ECM. Focal adhesion component–mediated signaling has been shown to regulate growth and migration. During cell migration, focal adhesions are formed toward the leading edge of cells whereas disassembly of adhesions occurs in the trailing edge of cells (22). This cycle of assembly and disassembly of focal adhesions is part of the process that allows the cell to move forward. These are important sites for the signaling events involved in regulation of different steps of cancer metastasis, including detachment, migration, invasion, extravasation, and proliferation of the cancer cells (42). Moreover, the transformation of epithelial cells into invasive carcinomas also depends on reorganization of the actin cytoskeleton that leads to stress fiber assembly (43). Our results suggest that Win55,212-2- and JWH-015–mediated effects on AKT, focal adhesion, and stress fiber may lead to reduced growth of NSCLC during metastasis. Furthermore, we believe that synthetic cannabinoids do not inhibit lung metastasis by inhibiting the attachment of the cells to lung tissue, as mice were treated 1 week after injection of cells. Therefore, synthetic cannabinoids might be inhibiting growth of these cells in lung tissue by inhibiting focal adhesions or signaling through Akt. CB1-mediated inhibition of stress fiber and focal adhesion formation in different cell types have previously been reported (44, 45).

Cannabinoid-treated tumors show a significantly reduced number of proliferating cells, impaired tumor vascularization, and an increase in apoptosis-mediated cell death. The antiproliferative and antiangiogenic effect of different cannabinoids has been observed previously in in vitro as well as in vivo studies (19, 46, 47). We have also shown that synthetic cannabinoids inhibit MMP-9 activity. MMP-9 has been shown to play an important role in growth and metastasis of various cancers by degrading ECM (26–28).

We show for the first time that CB1 and CB2 receptors are overexpressed in NSCLC patient tissue samples. Furthermore, nonpsychoactive small molecular weight synthetic cannabinoids inhibited growth, migration, and invasion of NSCLC cells in in vitro as well as in vivo in a mouse model. In addition, molecular mechanism of inhibition reveals synthetic cannabinoid agonists may inhibit tumor growth and metastasis by inhibiting AKT phosphorylation and formation of focal adhesion structures. Our results suggest the use of nonpsychoactive synthetic cannabinoid ligands as a promising strategy for inhibiting growth and metastasis of highly resistant NSCLC. Overall, our results indicate a novel role for cannabinoid receptors CB1 and CB2 in NSCLC growth and metastasis.

The authors declare that they have no potential conflicts of interest.

We thank Justine Curley Cohen and Wendy Dasgupta, Beth Israel Deaconess Medical Center microscopy core facility for staining and analysis of the lung histology microscopy specimens. We would also like to thank Susie Jones, Department of Pathology, The Ohio State University, for the immunostaining of TMA and tumor slides.

This work is partly supported by grants from the National Institutes of HealthR01CA109527 (R.K. Ganju).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Jemal
A
,
Siegel
R
,
Ward
E
,
Hao
Y
,
Xu
J
,
Thun
MJ
. 
Cancer statistics
, 
2009
.
CA
Cancer J Clin
2009
;
59
:
225
49
.
2.
Kotsakis
A
,
Georgoulias
V
. 
Targeting epidermal growth factor receptor in the treatment of non-small-cell lung cancer
.
Expert Opin Pharmacother
2010
;
11
:
2363
89
.
3.
Wheeler
DL
,
Dunn
EF
,
Harari
PM
. 
Understanding resistance to EGFR inhibitors-impact on future treatment strategies
.
Nat Rev Clin Oncol
2007
;
7
:
493
507
.
4.
Franklin
WA
,
Veve
R
,
Hirsch
FR
,
Helfrich
BA
,
Bunn
PA
 Jr
. 
Epidermal growth factor receptor family in lung cancer and premalignancy
.
Semin Oncol
2002
;
29
:
3
14
.
5.
Salomon
DS
,
Brandt
R
,
Ciardiello
F
,
Normanno
N
. 
Epidermal growth factor-related peptides and their receptors in human malignancies
.
Crit Rev Oncol Hematol
1995
;
19
:
183
232
.
6.
Ciardiello
F
. 
An update of new targets for cancer treatment: receptor-mediated signals
.
Ann Oncol
2002
;
13
Suppl 4
:
29
38
.
7.
Viloria-Petit
A
,
Crombet
T
,
Jothy
S
,
Hicklin
D
,
Bohlen
P
,
Schlaeppi
JM
, et al
Acquired resistance to the antitumor effect of epidermal growth factor receptor-blocking antibodies in vivo: a role for altered tumor angiogenesis
.
Cancer Res
2001
;
61
:
5090
101
.
8.
Matsuda
LA
,
Lolait
SJ
,
Brownstein
MJ
,
Young
AC
,
Bonner
TI
. 
Structure of a cannabinoid receptor and functional expression of the cloned cDNA
.
Nature
1990
;
346
:
561
4
.
9.
Munro
S
,
Thomas
KL
,
Abu-Shaar
M
. 
Molecular characterization of a peripheral receptor for cannabinoids
.
Nature
1993
;
365
:
61
5
.
10.
Guzman
M
. 
Cannabinoids: potential anticancer agents
.
Nat Rev Cancer
2003
;
3
:
745
55
.
11.
Qamri
Z
,
Preet
A
,
Nasser
MW
,
Bass
CE
,
Leone
G
,
Barsky
SH
, et al
Synthetic cannabinoid receptor agonists inhibit tumor growth and metastasis of breast cancer
.
Mol Cancer Ther
2009
;
8
:
3117
29
.
12.
Xu
X
,
Liu
Y
,
Huang
S
,
Liu
G
,
Xie
C
,
Zhou
J
, et al
Overexpression of cannabinoid receptors CB1 and CB2 correlates with improved prognosis of patients with hepatocellular carcinoma
.
Cancer Genet Cytogenet
2006
;
171
:
31
8
.
13.
Sarfaraz
S
,
Adhami
VM
,
Syed
DN
,
Afaq
F
,
Mukhtar
H
. 
Cannabinoids for cancer treatment: progress and promise
.
Cancer Res
2008
;
68
:
339
42
.
14.
Sarfaraz
S
,
Afaq
F
,
Adhami
VM
,
Malik
A
,
Mukhtar
H
. 
Cannabinoid receptor agonist-induced apoptosis of human prostate cancer cells LNCaP proceeds through sustained activation of ERK1/2 leading to G1 cell cycle arrest
.
J Biol Chem
2006
;
281
:
39480
91
.
15.
Caffarel
MM
,
Andradas
C
,
Mira
E
,
Pérez-Gómez
E
,
Cerutti
C
,
Moreno-Bueno
G
, et al
Cannabinoids reduce ErbB2-driven breast cancer progression through Akt inhibition
.
Mol Cancer
2010
;
9
:
196
.
16.
Mackie
K
. 
Cannabinoid receptors as therapeutic targets
.
Annu Rev Pharmacol Toxicol
2006
;
46
:
101
22
.
17.
Preet
A
,
Ganju
RK
,
Groopman
JE
. 
Delta9-Tetrahydrocannabinol inhibits epithelial growth factor-induced lung cancer cell migration in vitro as well as its growth and metastasis in vivo
.
Oncogene
2008
;
27
:
339
46
.
18.
Caffarel
MM
,
Sarrio
D
,
Palacios
J
,
Guzman
M
,
Sanchez
C
. 
Delta9-tetrahydrocannabinol inhibits cell cycle progression in human breast cancer cells through Cdc2 regulation
.
Cancer Res
2006
;
66
:
6615
21
.
19.
Casanova
ML
,
Blazquez
C
,
Martinez-Palacio
J
,
Villanueva
C
,
Fernández-Aceñero
MJ
,
Huffman
JW
, et al
Inhibition of skin tumor growth and angiogenesis in vivo by activation of cannabinoid receptors
.
J Clin Invest
2003
;
111
:
43
50
.
20.
Bhola
NE
,
Grandis
JR
. 
Crosstalk between G-protein-coupled receptors and epidermal growth factor receptor in cancer
.
Front Biosci
2008
;
13
:
1857
65
.
21.
Kishimoto
S
,
Gokoh
M
,
Oka
S
,
Muramatsu
M
,
Kajiwara
T
,
Waku
K
, et al
2-arachidonoylglycerol induces the migration of HL-60 cells differentiated into macrophage-like cells and human peripheral blood monocytes through the cannabinoid CB2 receptor-dependent mechanism
.
J Biol Chem
2003
;
278
:
24469
75
.
22.
Lauffenburger
DA
,
Horwitz
AF
. 
Cell migration: a physically integrated molecular process
.
Cell
1996
;
84
:
359
69
.
23.
Song
ZH
,
Zhong
M
. 
CB1 cannabinoid receptor-mediated cell migration
.
J Pharmacol Exp Ther
2000
;
294
:
204
9
.
24.
Ullrich
A
,
Schlessinger
J
. 
Signal transduction by receptors with tyrosine kinase activity
.
Cell
1990
;
61
:
203
12
.
25.
Yarden
Y
. 
The EGFR family and its ligands in human cancer. Signalling mechanisms and therapeutic opportunities
.
Eur J Cancer
2001
;
37
Suppl 4
:
S3
8
.
26.
Van Trappen
PO
,
Ryan
A
,
Carroll
M
,
Lecoeur
C
,
Goff
L
,
Gyselman
VG
, et al
A model for co-expression pattern analysis of genes implicated in angiogenesis and tumour cell invasion in cervical cancer
.
Br J Cancer
2002
;
87
:
537
44
.
27.
Hao
L
,
Zhang
C
,
Qiu
Y
, et al
Recombination of CXCR4, VEGF, and MMP-9 predicting lymph node metastasis in human breast cancer
.
Cancer Lett
2007
;
253
:
34
42
.
28.
Kumta
SM
,
Huang
L
,
Cheng
YY
,
Chow
LT
,
Lee
KM
,
Zheng
MH
. 
Expression of VEGF and MMP-9 in giant cell tumor of bone and other osteolytic lesions
.
Life Sci
2003
;
73
:
1427
36
.
29.
Breathnach
OS
,
Freidlin
B
,
Conley
B
,
Green
MR
,
Johnson
DH
,
Gandara
DR
, et al
Twenty-two years of phase III trials for patients with advanced non-small-cell lung cancer: sobering results
.
J Clin Oncol
2001
;
19
:
1734
42
.
30.
Kelly
K
,
Crowley
J
,
Bunn
PA
 Jr, Presant CA, Grevstad PK, Moinpour CM
, et al
Randomized phase III trial of paclitaxel plus carboplatin versus vinorelbine plus cisplatin in the treatment of patients with advanced non–small-cell lung cancer: a Southwest Oncology Group trial
.
J Clin Oncol
2001
;
19
:
3210
8
.
31.
Schiller
JH
,
Harrington
D
,
Belani
CP
,
Langer
C
,
Sandler
A
,
Krook
J
, et al
Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer
.
N Engl J Med
2002
;
346
:
92
8
.
32.
Giaccone
G
,
Herbst
RS
,
Manegold
C
,
Scagliotti
G
,
Rosell
R
,
Miller
V
, et al
Gefitinib in combination with gemcitabine and cisplatin in advanced non-small-cell lung cancer: a phase III trial—INTACT 1
.
J Clin Oncol
2004
;
22
:
777
84
.
33.
Herbst
RS
,
Giaccone
G
,
Schiller
JH
,
Natale
RB
,
Miller
V
,
Manegold
C
, et al
Gefitinib in combination with paclitaxel and carboplatin in advanced non-small-cell lung cancer: a phase III trial–INTACT 2
.
J Clin Oncol
2004
;
22
:
785
94
.
34.
Portella
G
,
Laezza
C
,
Laccetti
P
,
De Petrocellis
L
,
Di Marzo
V
,
Bifulco
M
. 
Inhibitory effects of cannabinoid CB1 receptor stimulation on tumor growth and metastatic spreading: actions on signals involved in angiogenesis and metastasis
.
FASEB J
2003
;
17
:
1771
3
.
35.
Gustafsson
K
,
Christensson
B
,
Sander
B
,
Flygare
J
. 
Cannabinoid receptor-mediated apoptosis induced by R(+)-methanandamide and Win55,212-2 is associated with ceramide accumulation and p38 activation in mantle cell lymphoma
.
Mol Pharmacol
2006
;
70
:
1612
20
.
36.
Sanchez
C
,
de Ceballos
ML
,
Gomez del Pulgar
T
,
Rueda
D
,
Corbacho
C
,
Velasco
G
, et al
Inhibition of glioma growth in vivo by selective activation of the CB(2) cannabinoid receptor
.
Cancer Res
2001
;
61
:
5784
9
.
37.
Ellert-Miklaszewska
A
,
Kaminska
B
,
Konarska
L
. 
Cannabinoids down-regulate PI3K/Akt and Erk signalling pathways and activate proapoptotic function of Bad protein
.
Cell Signal
2005
;
17
:
25
37
.
38.
White
SR
,
Tse
R
,
Marroquin
BA
. 
Stress-activated protein kinases mediate cell migration in human airway epithelial cells
.
Am J Respir Cell Mol Biol
2005
;
32
:
301
10
.
39.
Toker
A
,
Yoeli-Lerner
M
. 
Akt signaling and cancer: surviving but not moving on
.
Cancer Res
2006
;
66
:
3963
6
.
40.
Hauck
CR
,
Sieg
DJ
,
Hsia
DA
,
Loftus
JC
,
Gaarde
WA
,
Monia
BP
, et al
Inhibition of focal adhesion kinase expression or activity disrupts epidermal growth factor-stimulated signaling promoting the migration of invasive human carcinoma cells
.
Cancer Res
2001
;
61
:
7079
90
.
41.
Huang
C
,
Rajfur
Z
,
Borchers
C
,
Schaller
MD
,
Jacobson
K
. 
JNK phosphorylates paxillin and regulates cell migration
.
Nature
2003
;
424
:
219
23
.
42.
van
Nimwegen
MJ
,
Verkoeijen
S
,
van Buren
L
,
Burg
D
,
van de Water
B
. 
Requirement for focal adhesion kinase in the early phase of mammary adenocarcinoma lung metastasis formation
.
Cancer Res
2005
;
65
:
4698
706
.
43.
Friedl
P
,
Wolf
K
. 
Tumour-cell invasion and migration: diversity and escape mechanisms
.
Nat Rev Cancer
2003
;
3
:
362
74
.
44.
Kumar
A
,
Song
ZH
. 
CB1 cannabinoid receptor-mediated changes of trabecular meshwork cellular properties
.
Mol Vis
2006
;
12
:
290
7
.
45.
Zhou
D
,
Song
ZH
. 
CB1 cannabinoid receptor-mediated neurite remodeling in mouse neuroblastoma N1E-115 cells
.
J Neurosci Res
2001
;
65
:
346
53
.
46.
Blazquez
C
,
Casanova
ML
,
Planas
A
,
Gómez Del Pulgar
T
,
Villanueva
C
,
Fernández-Aceñero
MJ
, et al
Inhibition of tumor angiogenesis by cannabinoids
.
FASEB J
2003
;
17
:
529
31
.
47.
Johnson
DR
,
Stebulis
JA
,
Rossetti
RG
,
Burstein
SH
,
Zurier
RB
. 
Suppression of fibroblast metalloproteinases by ajulemic acid, a nonpsychoactive cannabinoid acid
.
J Cell Biochem
2007
;
100
:
184
90
.