Human ErbB2-positive breast tumors express CB₂ cannabinoid receptors

We first analyzed whether ErbB2-positive human breast tumors express cannabinoid targets (i.e. cannabinoid receptors). We performed an immunohistochemical analysis of CB₁ and CB₂ receptors in 87 grade 3 invasive breast ductal carcinomas and 6 non-tumoral mammary samples by tissue microarrays. CB₁ immunoreactivity was detected in only 14% of the tumors (12/87), and no correlation was found between this receptor expression and ErbB2 expression (p = 0.198, Fig. ​Fig.1).1). Conversely, CB₂ receptor staining was evident in 72% of the carcinomas (63/87) and it was significantly associated with ErbB2 expression, since it was observed in 91% of the ErbB2-positive tumors (21/23, p = 0.018, Fig. ​Fig.1).1). Moreover, we detected no significant CB₁ or CB₂receptor immunoreactivity in non-transformed mammary tissue (data not shown).

Figure 1

ErbB2-positive human breast tumors express cannabinoid receptors. (A) Representative images of human breast tumors negative (upper row) or positive (lower row) for CB₁, CB₂ and ErbB2 receptors (brown). Scale bars: 200 μm; insets: 100 μm. …

Cannabinoids exert an antitumoral effect in the MMTV-neu model of breast cancer

We then analyzed the effect of cannabinoids on tumor progression in a well established and clinically relevant animal model of ErbB2-driven metastatic breast cancer, the MMTV-neu mouse. We first observed that our MMTV-neu colony develops breast tumors after a long latency period similar to that previously reported [9]. In particular, 50% of the females had tumors by week 36 (Additional file 1: Fig. S1A). Overexpression of the rat ErbB2 transgene (neu) in the tumors was verified by real-time quantitative PCR (Additional file 1: Fig. S1B). Treatment with cannabinoids, either THC, the main marijuana-derived cannabinoid in terms of abundance and potency, or JWH-133, a synthetic CB₂receptor-selective agonist, strongly slowed down tumor growth (Fig. ​(Fig.2A),2A), leading to smaller lesions at the end of the treatment (Additional file 1: Fig. S2). These compounds, however, did not change the histomorphologic features of the tumors. Thus, the three different experimental groups generated focal, ductal, solid, well vascularized mammary tumors surrounded by a non-invasive hyperplasic mammary epithelium (Fig. ​(Fig.2B).2B). We also observed that MMTV-neu-derived tumors express CB₁ and CB₂ cannabinoid receptor mRNA (determined by real-time quantitative PCR, data not shown) and protein (Additional file 1: Fig. S3).

Figure 2

Cannabinoids inhibit breast tumor growth in vivo and the number of tumors generated per animal. (A) Volume time-course (scale bar: 1 cm) of the first tumor appeared in each animal. (B) Representative images (H&E staining) of the histopathology …

Of interest, cannabinoids not only impaired tumor growth, but also blocked tumor generation per se. Thus, while 41% of vehicle-treated animals developed 4 or more tumors (up to 6), cannabinoid-treated animals never developed more than 3 tumors (Fig. ​(Fig.2C,2C, p < 0.05). Consequently, total tumor burden was strikingly decreased by cannabinoids (Fig. ​(Fig.2D).2D). There was also a delay in the appearance of the subsequent tumors in these animals. Thus, the average latency for the generation of a second tumor in vehicle-treated, THC-treated and JWH-133-treated animals was 33, 46 and 54 days, respectively. As mentioned in the Methods section, only the first tumor in each animal was treated peritumorally with cannabinoids. However, we detected a remarkable growth-inhibitory effect of cannabinoids in those tumors appeared in second place (Fig. ​(Fig.2E2E).

Cannabinoids impact tumor cell proliferation, tumor cell survival and tumor angiogenesis

We next analyzed the proliferative potential of cancer cells and found that it was reduced by both THC and JWH-133, as indicated by a decreased number of Ki67-positive cells in cannabinoid-treated tumors (Fig. ​(Fig.3A).3A). Cannabinoid administration also increased the number of cleaved (active) caspase 3-positive cells within the tumors, indicating that these compounds induce cancer cell death by apoptosis (Fig. ​(Fig.3B).3B). Tumor vascularization was also impaired by cannabinoids, as both THC and JWH-133 decreased the number of blood vessels in the tumors, as determined by CD31 staining (Fig. ​(Fig.3C).3C). To evaluate the possible contribution of the immune response to cannabinoid antitumoral action, we analyzed by immunofluorescence the degree of immune infiltration in the tumors. The percentage of CD45-positive cells (differentiated hematopoietic cells except erythrocytes and platelets) within the tumors was very low in all the samples tested and no significant differences between experimental groups were detected (Fig. ​(Fig.3D).3D). These data suggest that cannabinoid treatment does not affect the infiltration of immune cells into the tumor parenchyma.

Figure 3

Cannabinoids inhibit cancer cell proliferation, induce cancer cell apoptosis, and impair tumor angiogenesis in vivo. (A) Ki67-positive cells (red), (B) active caspase-3-positive cells (red), (C) CD31-positive cells (green) and (D) CD45-positive area in …

Cannabinoids decrease breast cancer metastases in the lungs

It has been previously reported that a high percentage of tumor-bearing MMTV-neu animals develop metastases in the lungs [9]. Specifically, we detected lung metastases (Fig. ​(Fig.4A)4A) in 67% of our vehicle-treated MMTV-neu animals (Fig. ​(Fig.4B).4B). The cell morphology, tumor architecture, and overexpression of the neu transgene mRNA in these lung structures confirmed the metastatic nature of the lesions (Figs. S1C and D). THC reduced the percentage of animals with lung metastases (Fig. ​(Fig.4B).4B). Although JWH-133 did not decrease this proportion, it significantly reduced the magnitude of the lesions. Thus, half of the metastases in this experimental group were detectable only by microscopic analysis (Fig. ​(Fig.4B).4B). As it was observed for the primary breast tumors, cannabinoid treatment did not alter the histopathology of the metastases, and the three experimental groups presented similar solid adenocarcinomas (Additional file 1: Fig. S1C). No sign of metastasis was detected in any of the other organs analyzed (brain, spleen, liver, kidneys -by histological analysis- and bones -by X-rays) in any of the experimental groups (data not shown).

Figure 4

Cannabinoids inhibit breast cancer metastasis to the lungs in vivo. (A) Metastatic lung nodules (pointed by arrows). (B) Percentage of animals with lung metastases. Macroscopic metastases were visible to the naked eye and microscopic lesions were detectable …

Degradation of the extracellular matrix is a crucial step in the metastatic process, especially during tumor cell intravasation and extravasation [10]. Matrix metalloproteinases (MMPs) have long been associated with this process owing to their ability to degrade the components of the extracellular matrix. To analyze whether cannabinoid administration affects MMP activity we conducted gelatin zymographies. MMP2 activity was decreased in THC- and JWH-133-treated tumors, while MMP9 activity was enhanced by cannabinoid treatment (Fig. ​(Fig.4C).4C). This THC- and JWH-133-induced reduction in MMP2 activity was accompanied by a decrease in MMP2 mRNA levels (Additional file 1: Fig. S4A). Conversely, cannabinoids did not change the amount of MMP9 transcripts (Additional file 1: Fig. S4A) and enhanced its protein levels (Additional file 1: Fig. S4B), indicating that they regulate MMP9 post-transcriptionally.

Akt downregulation is involved in cannabinoid antitumoral action

We next aimed at characterizing the mechanism underlying cannabinoid antitumoral effect. It is well established that several types of human cancers are associated with deregulation of signaling via ErbB members [1]. In particular, ErbB2 overexpression correlates, for instance, with tumor size, increased metastatic potential, and higher histological grade, implying that ErbB2 confers a strong proliferative and survival advantage to tumor cells [11]. To assess whether cannabinoids modulate the expression of endogenous ErbB2 and of the rat ErbB2 ortologue neu, which is ectopically expressed in our animal model, we conducted real-time quantitative PCR determinations upon THC and JWH-133 treatment. However, no significant changes were detected (Fig. ​(Fig.5A5A).

Figure 5

THC inhibits Akt in vivo. (A) Mouse ErbB2 and (B) rat ErbB2 (neu transgene) mRNA expression in vehicle- and cannabinoid-treated tumors as determined by real-time quatitative PCR. (B and C) Western blot and densitometric analysis of phospho-Akt (B) and …

A central intracellular signaling pathway activated by ErbB2 is the PI3K/Akt pathway, whose importance in breast cancer is corroborated by clinical studies showing Akt activation in most ErbB2-overexpressing tumors [11]. We observed a decrease in Akt activation in THC-treated MMTV-neu tumors (Fig. ​(Fig.5B)5B) as well as diminished levels of phosphorylated S6 ribosomal protein [a read-out for activation of the Akt/mammalian target of rapamycin (mTOR) pathway [12]] (Fig. ​(Fig.5C).5C). To determine the importance of Akt inhibition in cannabinoid antitumoral action we conducted different experiments with the cell line N202.1A, that was isolated from a MMTV-neu breast tumor [13]. THC and JWH-133 decreased N202.1A cell proliferation (Fig. ​(Fig.6A)6A) in a CB₂ receptor-dependent manner, as indicated by the prevention of cannabinoid action exerted by the CB₂ receptor-selective antagonist SR144528 but not by the CB₁ receptor-selective antagonist SR141716 (Fig. ​(Fig.6B).6B). Likewise, the growth rate of N202.1A-derived xenografts was significantly diminished by THC and JWH-133, and this effect was prevented by SR144528 (Fig. ​(Fig.6C).6C). THC also decreased cell proliferation of two different ErbB2-overexpressing breast cancer cell lines of human origin (Additional file 1: Fig. S5), suggesting that human ErbB2-positive breast tumor cells may be sensitive to cannabinoid antitumoral action as well. N202.1A cells showed a dose-dependent reduction in Akt activation (Fig. ​(Fig.6D).6D). Of interest, overexpression of a myristoylated (i.e. constitutively activated) form of Akt (Fig. ​(Fig.6E)6E) prevented THC antiproliferative effect (Fig. ​(Fig.6F).6F). To further support the importance of Akt in cannabinoid antitumoral action, subcutaneous xenografts were generated in nude mice with N202.1A cells stably expressing myristoylated Akt or the corresponding empty vector (pBABE). As shown in Fig. ​Fig.6G6G (left panel), THC significantly reduced the growth of pBABE-transfected N202.1A-derived tumors. In contrast, overexpression of activated Akt prevented THC effect on tumor progression (Fig. ​(Fig.6G,6G, right panel). The same effect was observed with JWH-133 (Fig. ​(Fig.6H6H).

Figure 6

Akt downregulation is involved in cannabinoid antitumoral action. (A and B) Viability of N202.1A cells in response to (A) increasing concentrations of THC or JWH-133 or (B) 6 μM THC or 10 μM JWH-133 with or without 2 μM SR141716 …

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-CB₁ receptor antibody was generously donated by Dr. Ken Mackie, Indiana University, Indiana, and anti-CB₂ 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.

Metastasis detection

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 CB₁receptor, 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.

Figures S1-S5. Supplemental Figure 1: MMTV-neu mice spontaneously develop breast tumors and lung metastases. Supplemental Figure 2: Cannabinoids inhibit breast tumor growth in vivo Supplemental Figure 3: MMTV-neu-derived tumors express cannabinoid receptors Supplemental Figure 4: Cannabinoids modulate the expression of MMP2 and MMP9 Supplemental Figure 5: Human ErbB2-positive breast cancer cell lines are sensitive to cannabinoids

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