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 . 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) . 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 . 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 . 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 .
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 . 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 . 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  and that the loss of CB1 receptors accelerates intestinal adenoma growth , 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 . 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 .
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 . 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. ], clinical studies have established a correlation between MMP9 overexpression and good prognosis in breast cancer . This protective effect might derive from its capacity to generate angiogenesis inhibitors such as angiostatin and tumstatin . The inhibition of MMP2 by cannabinoids shown here, in line with that previously reported by Bifulco and coworkers in thyroid cancer cells  and our group in gliomas , may be of special relevance considering that high tumor levels of this metalloproteinase have been correlated with poor prognosis in breast cancer . In addition, enhanced levels of MMP2 in breast tumors are associated with ErbB2 gene amplification and/or overexpression . Moreover, Massagué and coworkers have recently identified MMP2 as one of the genes of the signature that mediates breast cancer metastasis to the lungs , 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 CB1 receptors [5, 6], one reasonable approach would be targeting CB2 receptors selectively. Here, we have demonstrated that the CB2-selective agonist JWH-133 is as effective as THC (a CB1/CB2-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 CB2 receptors, and (ii) that a very low fraction of them express CB1 receptors. Taken together, these data suggest that activation of CB2 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 CB2 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 CB2 levels than steroid receptor-positive lesions . Moreover, ErbB2-positive tumors also have increased CB2 receptor mRNA levels compared to their less aggressive and more responsive ErbB2-negative counterparts . Of interest, this receptor is scarcely expressed in non-transformed mammary tissue [data presented here and [17, 21]].