Background
Breast cancer represents approximately 30% of newly diagnosed cancers each year. Almost one third of them overexpresses the ErbB2 tyrosine kinase receptor (Her2 in humans, Neu in rats), a member of the EGF receptor family [
1]. Phosphorylation of their intracellular domains upon engagement by their ligands induce receptor homo- or heterodimerization, leading to the activation of key signaling pathways that promote cell proliferation and survival, including the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and the ERK/MAPK cascade. Although no specific ligand for ErbB2 has been identified yet, this receptor is the preferred heterodimerization partner of the family [
2]. ErbB2-overexpressing breast tumors are characterized by very aggressive clinical courses and decreased survival rates, mostly due to the poorly differentiated, highly proliferative and highly invasive nature of their constituent cells [
2]. All these characteristics make ErbB2-overexpressing tumors less responsive to conventional therapies. One of the most recent advances in the treatment of these tumors is the use of a humanized neutralizing monoclonal antibody against ErbB2 (Trastuzumab) [
3]. Although this strategy has been very successful, around 75% of patients with ErbB2-overexpressing tumors do not respond to Trastuzumab, and nearly 15% of the responders eventually develop metastases [
4]. The existence of this considerable population of non-responding and relapsing patients urges the search for novel treatments.
The therapeutic potential of cannabinoids, the active compounds of marijuana and their derivatives, has been known for centuries. There is increasing evidence supporting that they might be beneficial in various pathological contexts such as pain, inflammation, eating disorders, and brain damage, amongst others [
5,
6]. Cannabinoids exert most of their actions by binding to and activating specific G protein-coupled receptors. To date, two cannabinoid receptors, namely CB
1 and CB
2, have been cloned and characterized from mammalian tissues, the main difference between them being their tissue expression pattern. Thus, while CB
1 receptors are ubiquitously located, with their highest presence found in the central nervous system, CB
2 receptor expression is mostly restricted to particular elements of the immune system [
5,
6]. During the last decade, evidence has accumulated suggesting that cannabinoids might be useful for the treatment of cancer. These compounds exert anti-proliferative, pro-apoptotic, anti-angiogenic, and anti-invasive effects in different cell-culture and animal models of cancer [
7,
8]. Here, we used a genetically engineered animal model of ErbB2-driven metastatic breast cancer (the MMTV-neu mouse) to analyze the antitumoral potential of cannabinoids in this particularly aggressive pathology. These animals express the rat ErbB2 oncogene (neu) under the control of the hormone-sensitive mouse mammary tumor virus-long terminal repeat (MMTV-LTR) promoter [
9]. Selective overexpression of neu in the mammary epithelium results in the spontaneous development of focal mammary tumors after a long latency (5-12 months) [
9]. Results presented herein (i) show that ErbB2-positive invasive human breast tumors express CB
2 receptors, (ii) demonstrate that Δ
9-tetrahydrocannabinol (THC) and the non-psychotropic CB
2 cannabinoid receptor agonist JWH-133 significantly reduce tumor progression in a clinically relevant model of ErbB2-positive metastatic breast cancer, and (iii) shed light on the mechanism of cannabinoid antitumoral action
in vivo.
Discussion
Aberrant ErbB2 expression and/or function yield highly aggressive tumors, with increased resistance to conventional chemotherapies and poor outcomes. Although the use of the anti-ErbB2 monoclonal antibody Trastuzumab markedly improves the survival of these patients, only 25% of them respond to this treatment and most of the responders eventually relapse [
14]. Moreover, the use of this antibody has been associated with important cardiotoxic side effects (severe congestive heart failure and decrease in left ventricular ejection fraction) [
14]. Consequently, extensive efforts should be made to find novel agents for the treatment of ErbB2-positive breast tumors. Our results demonstrate that, in spontaneously aroused ErbB2-overexpressing breast tumors, cannabinoids inhibit tumor generation, growth, vascularization, and metastasis. Although a cannabinoid-based monotherapy might be potentially effective for ErbB2-positive breast tumors, it would be interesting to analyze the effect of these compounds in combination with other anticancer treatments. Thus, it is worth noting that Trastuzumab, the most relevant targeted therapy for ErbB2-positive tumors so far, has a modest median overall response when used as a first-line agent, an efficacy that is clearly enhanced when used in combination with other chemotherapeutic agents [
14]. Additionally, Akt overactivation has been detected in a significant percentage of primary human breast cancers, in which it is associated to enhanced resistance to Trastuzumab [
14,
15]. Our results show that downregulation of Akt is involved in cannabinoid antitumoral action. This kinase is the central node of the PI3K/Akt/mTOR signaling pathway, that activates crucial processes such as cell survival, cell growth, cell proliferation, angiogenesis, and cell migration and invasion [
12]. This pathway is therefore an attractive target for anticancer agents and, as a matter of fact, clinical trials have been/are being conducted with mTOR, PI3K and Akt inhibitors [
3].
The antitumoral potential of cannabinoids has been documented both
in vitro and in animal models of cancer [
7,
8]. These compounds inhibit breast cancer cell proliferation
in vitro through processes that include cell cycle arrest [
16‐
21], hormone and growth-factor receptor modulation [
18,
22,
23], and apoptosis induction [
17,
20,
21]. The
in vivo approaches followed so far have been mostly based on xenograft models [
20,
21], which are helpful but limited tools. These models rely on the propagation of cancer cell lines in immunodeficient mice at ectopic or orthotopic sites and lack crucial features of patients' tumors such as the actual tumor architecture and the interactions with the tumor microenvironment (including non-cancerous surrounding tissue, vasculature and immune cells) and diminished genetic heterogeneity [
24]. In contrast to xenografted animals, in the mutant mice used in this study tumors appear spontaneously and after long latency periods, recruit and generate blood vessels, and penetrate the vasculature giving rise to distant metastases [
9]. These features parallel the human pathology much more closely and make the MMTV-neu mice a clinically relevant model of ErbB2-driven breast cancer.
Remarkably, this is, to the best of our knowledge, the first report supporting that cannabinoids hamper not only tumor growth but also tumor generation. Recently, Qamri and coworkers and DuBois and coworkers, by using two different genetic models of cancer, demonstrated that JWH-133 delays the appearance of breast tumors [
21] and that the loss of CB
1 receptors accelerates intestinal adenoma growth [
25], respectively, and Izzo
et al. observed that high endocannabinoid levels and the cannabinoid agonist HU-210 reduce the development of precancerous lesions in the mouse colon [
26]. These and our data suggest that the endocannabinoid system has a physiological protective role against tumorigenesis, in line with the general idea that this system contributes to maintain homeostasis in health and disease [
6].
Data presented herein show that cannabinoids modulate MMP activity. In particular, we report an inhibition of MMP2 and an activation of MMP9 by cannabinoids. Although MMPs have been traditionally associated to metastasis due to their ability to degrade the extracellular matrix, it has been recently shown that several members of this family provide a protective effect in different stages of cancer progression [
27]. One of these antitumoral MMPs is MMP9, which is activated by cannabinoids in our system. Although this MMP may promote the angiogenic switch in some experimental tumors [e.g. [
28]], clinical studies have established a correlation between MMP9 overexpression and good prognosis in breast cancer [
29]. This protective effect might derive from its capacity to generate angiogenesis inhibitors such as angiostatin and tumstatin [
27]. The inhibition of MMP2 by cannabinoids shown here, in line with that previously reported by Bifulco and coworkers in thyroid cancer cells [
30] and our group in gliomas [
31], may be of special relevance considering that high tumor levels of this metalloproteinase have been correlated with poor prognosis in breast cancer [
32]. In addition, enhanced levels of MMP2 in breast tumors are associated with ErbB2 gene amplification and/or overexpression [
33]. Moreover, Massagué and coworkers have recently identified MMP2 as one of the genes of the signature that mediates breast cancer metastasis to the lungs [
34], the targeted metastatic organ in our animal model.
Potential antitumoral therapies based on the use of cannabinoids might be limited by their well known psychotropic actions such as dizziness, dry mouth, tiredness, muscle weakness, euphoria, myalgia and palpitations [
6,
35]. Although the benefit/risk ratio is potentially high for cannabinoid-based therapies, different strategies should be taken to avoid or at least minimize their side effects. Since most -if not all- of the psychoactive effects of cannabinoids are produced by the activation of central CB
1 receptors [
5,
6], one reasonable approach would be targeting CB
2 receptors selectively. Here, we have demonstrated that the CB
2-selective agonist JWH-133 is as effective as THC (a CB
1/CB
2-mixed agonist) in reducing tumor generation and progression. Moreover, our results also (i) show that an elevated percentage of high grade ErbB2-positive human breast tumors express CB
2 receptors, and (ii) that a very low fraction of them express CB
1 receptors. Taken together, these data suggest that activation of CB
2 in this particular population of patients would be an efficient strategy to treat breast tumors without triggering psychoactive effects. A correlation between tumor aggressiveness and CB
2 receptor expression in breast cancer has been previously reported: tumors lacking estrogen or progesterone receptors, which are associated to low response rates to adjuvant therapies, express higher CB
2 levels than steroid receptor-positive lesions [
17]. Moreover, ErbB2-positive tumors also have increased CB
2 receptor mRNA levels compared to their less aggressive and more responsive ErbB2-negative counterparts [
17]. Of interest, this receptor is scarcely expressed in non-transformed mammary tissue [data presented here and [
17,
21]].
Methods
Tissue microarray analysis
Eighty seven grade 3 invasive breast ductal carcinomas and 6 non-tumoral mammary tissues were fixed in 10% paraformaldehide (PFA) and embedded in paraffin. Two representative tissue cores (1 mm of diameter) of each one were included in a tissue microarray. The main clinical pathology and molecular features of this series had been previously reported [
36]. Tissue sections were subjected to a heat-induced antigen retrieval step prior to exposure to the primary antibody. Anti-CB
1 receptor antibody was generously donated by Dr. Ken Mackie, Indiana University, Indiana, and anti-CB
2 receptor antibody was from Affinity Bioreagents/Thermo Fisher Scientific, Rockford, Illinois. ErbB2 expression was evaluated using a HercepTest (Dako, Carpenteria, CA) according to manufacturer's instructions. Immunodetection was performed using the LSAB method (DAKO) with DAB as the chromogen. In negative controls, the primary antibody was omitted or replaced with an irrelevant antibody. Cases were reviewed by two independent pathologists (J.P. and G.M-B) and were scored as positive for cannabinoid receptors when more than 25% of the neoplastic cell showed intense immunostaining for the corresponding antibody. ErbB2-staining was scored according to HercepTest manufacturer's guidelines: scores 0 and 1+ were considered as negative, and 2+ and 3+ as positive for ErbB2 overexpression.
Animals and treatments
All procedures involving animals were performed with the approval of the Complutense University Animal Experimentation Committee according to the European official regulations. FVB/N-Tg(MMTVneu)202 Mul/J mice (more commonly designated as MMTV-neu mice) were obtained from The Jackson Laboratory (Bar Harbor, Maine). Females were palpated twice weekly for mammary gland nodules and cannabinoid treatment was started when the first tumor in each animal was detected. Δ9-Tetrahydrocannabinol (THC, The Health Concept, Richelbach, Germany) and JWH-133 (kindly donated by John W. Huffman, Clemson University, South Carolina) were prepared in DMSO (0.2 mg/μL and 0.02 mg/μL, respectively) and diluted in PBS supplemented with 5% BSA (100 μL/dose for tumors ≤ 1000 mm3 and 200 μl/dose for tumors >1000 mm3). Cannabinoid peritumoral treatment (0.5 mg THC or 0.05 mg JWH-133/animal/day, twice a week) was maintained for 90 days, and only the first tumor in each animal was treated. Tumors were routinely measured during this period with external caliper, and volume was calculated as (4π/3) × (width/2)2 × (length/2). At the end of the treatment, animals were sacrificed and tumors and organs were collected. Tumors were divided in four portions for 1) preparation of tissue sections for immunofluorescent staining [frozen in Tissue-Tek (Sakura Finetek Europe, Zoeterwoude, The Netherlands)], 2) preparation of tissue sections for hematoxylin-eosin staining (fixed in buffered 4% PFA), 3) protein extraction (snap frozen) and 4) RNA isolation (snap frozen), and were stored at -80°C until analysis (except PFA-fixed tumor fractions, that were kept at room temperature). Brain, spleen, liver, kidneys and lungs were fixed in PFA. For xenograft experiments, subcutaneous tumors were induced in 6 week-old athymic female mice (Harlan Interfauna Iberica, Barcelona, Spain) by subcutaneous injection of 5 × 105 N202.1A cells. When tumors reached ca. 100 mm3, they were treated with THC (0.5 mg/animal/day), JWH-133 (50 μg/animal/day), SR144528 (50 μg/animal/day), a combination of cannabinoid and SR144528 or vehicle for 3 weeks, 3 times a week, measured, and processed as described above. For Akt-related experiments, half of the animals were injected with N202.1A cells stably expressing myristoylated Akt (N202.1A-pBABE-myr-Akt), and the other half with N202.1A cells stably expressing the corresponding empty vector (N202.1A-pBABE). Tumors were treated with THC, JWH-133 or vehicle and processed as described for N202.1A xenografts.
Collected organs were visually analyzed for macroscopic metastases. Microscopic metastases were determined by histological analysis of PFA-fixed paraffin-embedded hematoxylin-eosin stained sections. Radiographs were taken to evaluate the presence of bone metastases using a conventional X-ray equipment (Diagnost 93, Philips Medical Systems, Eindhoven, The Netherlands), and mammography cassette (Kodak MIN-R 2000 screen cassette) and film (Kodak MIN-R S film) (Eastman Kodak Company, Rochester, New York).
Cell culture and viability
N202.1A cells were kindly given by Dr. Vincenzo Bronte (Istituto Oncologico Veneto, Padova, Italy). This cell line was established from a MMTV-neu-derived tumor [
13]. BT474, MDA-MB-231, MCF-7 and SkBr3 human breast cancer cells, Jurkat human leukemic cells, and U373 human glioblastoma cells were from ATCC-LGC (Barcelona, Spain). All cell lines were maintained in DMEM supplemented with 10% fetal bovine serum (FBS). Cells were transferred to a low (0.5%)-FBS medium immediately before cannabinoid challenge. Cell viability was determined by the 3-4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide thiazol blue test (Sigma, St. Louis, Missouri) according to manufacturer's instructions.
Plasmids, transfections and infections
Stable expression of myr-Akt was achieved by retroviral infection. N202.1A cells were transduced for 4 h with supernatants obtained from Phoenix ecotropic cells previously transfected with a retroviral vector carrying HA-tagged myr-Akt (kindly provided by Dr. Pier P. Pandolfi, Harvard University, Boston, Massachusetts) or the corresponding empty construction (pBABE). Infected cells were selected with puromycin.
Immunofluorescence analysis
Tissue-tek embedded tumor sections were fixed in PFA and incubated with anti-CB1 receptor, anti-CB2 receptor, anti-CD31 (Pharmingen/BD Biosciences, San Jose, California), anti-CD45 (Pharmingen/BD Biosciences), anti-Ki67 (Neomarkers/Lab Vision, Fremont, California) or anti-cleaved-caspase 3 (Cell Signaling Technology, Danvers, Massachusetts) antibodies. Secondary anti-rabbit antibodies AlexaFluor 594 and AlexaFluor 488 were from Invitrogen (Carlsbad, California). Cell nuclei were stained with Hoescht 33342 (Invitrogen). Fluorescence images were acquired using Metamorph Premier Offline software (Molecular Devices, Sunnyvale, California). Blood vessel size was calculated with ImageJ software.
Real-time quantitative PCR (RTQ-PCR) and reverse-transcriptase PCR (RT-PCR)
RNA was isolated with Trizol Reagent (Invitrogen), including a DNase digestion step, with the Real Star Kit (Durviz, Valencia, Spain), and cDNA was obtained with Transcriptor Reverse Transcriptase (Roche Applied Science, Penzberg, Germany). The primers used for RTQ-PCR amplification were: mouse CB1 receptor, sense 5'-GGGCAAATTTCCTTGTAGCA-3', antisense 5'-GGCTCAACGTGACTGAGAAA-3'; mouse CB2 receptor, sense 5'-ATTCAGGAGATCTGTTAAGACAAGG-3', antisense 5'-GACATCTATGAAGTTGAGGCAGTG-3'; mouse MMP2, sense 5'- GCGCTTTTCTCGAATCCAT-3', antisense 5'-GGGTATCCATCTCCATGCTC-3'; mouse MMP9, sense 5'-ACGACATAGACGGCATCCA-3', antisense 5'- GCTGTGGTTCAGTTGTGGTG-3'; rat ErbB2 (neu), sense 5'-GCTCAGAGACCTGCTTTGGA-3', antisense 5'-AGGAGGACGAGTCCTTGTAGTG-3'; mouse ErbB2, sense 5'-AACAGCTCGGAGACCTGCTA-3', antisense 5'-GTAGTGGGCACAAGCCTCA-3'. Probes were from the Universal Probe Library (Roche Applied Science). Multispecies 18S RNA was used as reference (sense 5'- GCTCTAGAATTACCACAGTTATCCAA-3', antisense 5'- AAATCAGTTATGGTTCCTTTGGTC-3'). The primers use for RT-PCR were: mouse MMP2, sense 5'- TCTGCGATGAGCTTAGGGAAAC-3', antisense 5'-GACATACATCTTTGCAGGAGACAAG-3'; mouse MMP9, sense 5'-GGACGACGTGGGCTACGT-3', antisense 5'- CACGGTTGAAGCAAAGAAGGA-3'. GAPDH was used as reference (sense 5'-GGGAAGCTCACTGGCATGGCCTTCC-3', antisense 5'-CATGTGGGCCATGAGGTCCACCAC-3').
Western blot analysis
Cell lysates from tumors and cell lines were subjected to SDS-PAGE, and proteins transferred onto polyvinylidene fluoride membranes. Blots were incubated with the following antibodiess: anti-CB1 receptor, anti-CB2 receptor (Affinity Bioreagents), anti-MMP9 (Chemicon International INC, Temecula, California), anti-ErbB2 (Santa Cruz Biotechnology, Santa Cruz, California), anti-phospho-Akt (Ser473), anti-Akt, anti-phospho-S6 ribosomal protein (Cell Signaling) and anti-α-tubulin (Sigma). Luminograms were obtained with the Amersham Enhanced Chemiluminescence Detection Kit (GE Healthcare, Uppsala, Sweden) and densitometric analysis was performed with Quantity One software (Bio-Rad).
MMP activity assay
MMP2 (Gelatinase A) and MMP9 (Gelatinase B) activities were determined by gelatin zymography. Briefly, SDS-PAGE were run in the presence of 0.1% gelatin, washed with a 2.5% Triton X-100 containing buffer, and incubated overnight at 37°C in 50 mM Tris pH 7.5, 150 mM NaCl, 10 mM CaCl2, 0.1% Triton X100. Gels were then stained with Coomasie Blue and digested bands quantified by densitometric analysis as described above.
Statistical analysis
ANOVA with a post hoc analysis by the Student-Newman-Keuls' test was routinely used. For the analysis of metastases and the number of tumors per animal, a Pearson Х2 test was used. To determine the correlation between immunohistochemical (CB1 and CB2 expression) and clinical pathology (ErbB2) data, the Х2 test with Yates correction, or Fisher's exact test, was used. The SPSS for Windows program (SPSS, Inc., Chicago, IL, version 17.0) was used for this analysis. All P-values were two-sided. Unless otherwise stated, data are expressed as mean ± s.e.m.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MMC participated in the design of the experiments and carried out animal treatments, sample collection and processing (MMP activity assays, quantitative PCRs, Western blot analyses, immunofluorescence experiments and cell culture approaches) and statistical analyses. CA and EP-G participated in animal treatments, sample collection, quantitative PCRs and Western blot analyses. EM participated in the design of the study and carried out immunofluorescence experiments. CC participated in animal treatments and immunofluorescence experiments. JMF carried out the histopathological analyses of animal tumors and metastases. GM-B performed and analyzed the tissue microarrays of human samples. IG-R analyzed metastases in MMTV-neu mice by magnetic resonance imaging. JP carried out the histopathological characterization of human breast samples. SM participated in the design of the study. MG contributed to the conception and design of the study and critically revised the manuscript. CS conceived the study, elaborated its design, coordinated the research and wrote the manuscript. All authors read and approved the final manuscript.