Human ErbB2-positive breast tumors express CB 2 cannabinoid receptors

We first analyzed whether ErbB2-positive human breast tumors express cannabinoid targets (i.e. cannabinoid receptors). We performed an immunohistochemical analysis of CB 1 and CB 2 receptors in 87 grade 3 invasive breast ductal carcinomas and 6 non-tumoral mammary samples by tissue microarrays. CB 1 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. 1). Conversely, CB 2 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. 1). Moreover, we detected no significant CB 1 or CB 2 receptor 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 CB 1 , CB 2 and ErbB2 receptors (brown). Scale bars: 200 μm; insets: 100 μm. (B) Percentage of tumors scored as positive or negative for cannabinoid receptor expression amongst the ErbB2-negative (n = 64) and ErbB2-positive (n = 23) populations. Full size image

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 CB 2 receptor-selective agonist, strongly slowed down tumor growth (Fig. 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. 2B). We also observed that MMTV-neu-derived tumors express CB 1 and CB 2 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 the MMTV-neu-derived mammary tumors. Scale bars (from left to right): 200 μm, 100 μm and 50 μm. (C) Percentage of animals with 1, 2, 3, 4 or more tumors at the end of the treatment (90 days) in each experimental group. (D) Total tumor burden (total tumor volume per animal) determined 90 days after cannabinoid or vehicle treatment. (E) Volume of the tumors appeared in second, third or subsequent positions, 40 days after their appearance. The small size of the cannabinoid-treated groups is due to the very few second or third tumors appeared early enough to last 40 days in the animals before the end of the treatment (90 days after the appearance of the first tumor). Full size image

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. 2C, p < 0.05). Consequently, total tumor burden was strikingly decreased by cannabinoids (Fig. 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. 2E).

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. 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. 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. 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. 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 the tumors. Scale bars: A, 60 μm; B, 40 μm; C and D, 100 μm. Cell nuclei are in blue. Quantifications of Ki67-positive cells (A), active caspase-3-positive cells (B), the number of blood vessels (C) and CD45-positive area (D) in the tumors are shown in the corresponding graphs. Full size image

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. 4A) in 67% of our vehicle-treated MMTV-neu animals (Fig. 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. 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. 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 only by H&E staining. These latter lesions were found only in JWH-133-treated animals. (C) Gelatin zymographies of vehicle- and cannabinoid-treated tumors. Four representative tumors are shown per experimental group. PC: positive control (conditioned medium of H71080 cells stimulated with the phorbol ester PMA). Arrows point to the latent (pro-MMP) and active MMP bands according to the positive control and the expected MMP molecular weights. Non-contiguous parts of the same gel are shown. Graphs show the densitometric analysis of MMP2 and MMP9 activities. Data are expressed in arbitrary units. *, p < 0.05 vs vehicle-treated tumors; §, p = 0.068 vs vehicle-treated tumors. Full size image

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. 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. 5A).

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 phospho-S6 ribosomal protein (C) levels in MMTV-neu-derived tumors treated with vehicle or THC. Eight representative tumors are shown. Total Akt (B) and α-tubulin (C) were used for normalization. Non-contiguous parts of the same gel are shown. Optical densities are expressed in arbitrary units. Full size image

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. 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. 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. 6A) in a CB 2 receptor-dependent manner, as indicated by the prevention of cannabinoid action exerted by the CB 2 receptor-selective antagonist SR144528 but not by the CB 1 receptor-selective antagonist SR141716 (Fig. 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. 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. 6D). Of interest, overexpression of a myristoylated (i.e. constitutively activated) form of Akt (Fig. 6E) prevented THC antiproliferative effect (Fig. 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. 6G (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. 6G, right panel). The same effect was observed with JWH-133 (Fig. 6H).