Anticancer mechanisms of cannabinoids

G. Velasco phd,*†‡ C. Sánchez phd,*§ and M. Guzmán phd*†||
ABSTRACT
In addition to the well-known palliative effects of cannabinoids on some cancer-associated symptoms, a large body of evidence shows that these molecules can decrease tumour growth in animal models of cancer. They do so by modulating key cell signalling pathways involved in the control of cancer cell proliferation and survival. In addition, cannabinoids inhibit angiogenesis and decrease metastasis in various tumour types in laboratory animals. In this review, we discuss the current understanding of cannabinoids as antitumour agents, focusing on recent discover- ies about their molecular mechanisms of action, including resistance mechanisms and opportunities for their use in combination therapy. Those observations have already contributed to the foundation for the development of the rst clinical studies that will analyze the safety and potential clinical bene t of cannabinoids as anticancer agents.
Key Words Cannabinoids, apoptosis, autophagy, cell proliferation, angiogenesis, cell signalling, combination therapy Curr Oncol. 2016 Mar;23(S2):S23-S32 www.current-oncology.com
INTRODUCTION
Preparations of Cannabis sativa L. (marijuana) have been used for many centuries both medicinally and recreation- ally. However, the chemical structures of their unique ac- tive components—the cannabinoids—were not elucidated until the 1960s. Three decades later, the rst solid clues to cannabinoid molecular action were established, which led to an impressive expansion of basic cannabinoid research and a renaissance in the study of the therapeutic effects of cannabinoids in various elds, including oncology. Today, it is widely accepted that, of the approximately 108 can- nabinoids produced by C. sativa, Δ9-tetrahydrocannabinol (thc) is the most relevant because of its high potency and abundance in plant preparations1,2.
Tetrahydrocannabinol exerts a wide variety of bio- logic effects by mimicking endogenous substances—the endocannabinoids anandamide3 and 2-arachidonoylg- lycerol4,5—that engage speci c cell-surface cannabinoid receptors6. So far, two major cannabinoid-speci c recep- tors—cb1 and cb2—have been cloned from mammalian tissues and characterized7,8. In addition, other receptors such as the transient receptor potential cation channel subfamily V, member 1, and the orphan G protein–coupled receptor 55 have been proposed to act as endocannabinoid receptors6. Most of the effects produced by cannabinoids in the nervous system and in non-neural tissues rely on cb1 receptor activation. In contrast, the cb2 receptor was initially described to be present in the immune system6, but was more recently shown to also be expressed in cells
from other origins9,10. Notably, expression of the cb1 and cb2 receptors has been found in many types of cancer cells, but not necessarily correlating with the expression of those receptors in the tissue of origin9,11,12.
The endocannabinoids, together with their receptors and the proteins responsible for their synthesis, transport, and degradation, constitute the endocannabinoid system. Aside from its pivotal neuromodulatory activity13, the en- docannabinoid system exerts other regulatory functions in the body such as control of cardiovascular tone, energy metabolism, immunity, and reproduction14,15. This miscel- laneous activity makes the pharmacologic manipulation of the endocannabinoid system a promising strategy for the management of many diseases. Speci cally, cannabi- noids are well known to exert palliative effects in cancer patients14,15. Their best-established use is the inhibition of chemotherapy-induced nausea and vomiting15,16. Today, capsules of thc (Marinol: AbbVie, North Chicago, IL, U.S.A.) and its synthetic analogue nabilone (Cesamet: Meda Phar- maceuticals, Somerset, NJ, U.S.A.) are approved for that purpose. Cannabinoids also inhibit pain, and thus a stan- dardized cannabis extract (Sativex: GW Pharmaceuticals, Salisbury, U.K.) has already been approved in Canada and is currently the subject of large-scale phase iii clinical trials for managing cancer-associated pain. Another potential palliative effect of cannabinoids in oncology, supported by phase iii clinical trials, includes appetite stimulation and attenuation of wasting. In that respect, Marinol can currently be prescribed for anorexia associated with weight loss in aids patients.
Correspondence to: Guillermo Velasco, Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, c/ José Antonio Novais 12, Madrid 28040 Spain.
E-mail: gvelasco@ucm.es n DOI: http://dx.doi.org/10.3747/co.23.3080
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ANTICANCER MECHANISMS OF CANNABINOIDS, Velasco et al.
The therapeutic potential of cannabinoids in oncology might not be restricted to their aforementioned palliative actions. Thus, numerous studies have provided evidence that thc and other cannabinoids exhibit antitumour effects in a wide array of animal models of cancer12,16,17. More- over, those observations led to the development of several clinical studies to investigate the antitumour activity of thc in humans (see “Clinical Antitumour Effects of Can- nabinoids” later in this article). Nonetheless, a few studies have shown that, under certain conditions, cannabinoid treatment can stimulate cancer cell proliferation in vitro18,19 and interfere with the tumour-suppressor role of the im- mune system20,21. Likewise, reports about the role of the endocannabinoid system in cancer (tumour-suppressor or oncogenic) are con icting22.
Reports concerning the biologic role of the endocan- nabinoid system in cancer physiopathology are sparse. Although some exceptions that might be tumour-type- specific are known, cannabinoid receptors and their endogenous ligands are both generally upregulated in tumour tissue compared with non-tumour tissue16,22–24. Additionally, various studies have associated the expres- sion levels of cannabinoid receptors, endocannabinoids, or endocannabinoid-metabolizing enzymes with tumour ag- gressiveness22,25,26, which suggests that the endocannabi- noid system might be overactivated in cancer and hence pro-tumourigenic22. In support of that hypothesis, reports showed that genetic ablation of cb1 and cb2 receptors in mouse models of cancer reduces ultraviolet light–induced skin carcinogenesis27, that overexpression of cb2 receptor enhances the predisposition to leukemia after leukemia viral infection28, and that cb2 promotes her2 (human epi- dermal growth factor receptor 2) pro-oncogenic signalling in breast cancer29.
Conversely, and in line with the evidence supporting the hypothesis that pharmacologic activation of canna- binoid receptors decreases tumour growth12,16, upregula- tion of endocannabinoid-degrading enzymes has been observed in aggressive human tumours and cancer cell lines25,26, indicating that endocannabinoid signalling can also have a tumour-suppressive role. In support of that observation, deletion of cb1 receptors was noted to accelerate intestinal tumour growth in a genetic mouse model of colon cancer30, increased endocannabinoid levels were observed to diminish azoxymethane-induced precancerous lesions in the mouse colon31, and reduction in the expression of the endocannabinoid-degrading enzyme monoacylglycerol lipase was seen to decrease tumour growth in xenografted mice25.
Further studies—including analyses of the activa- tion of the precise signalling mechanisms involved in the regulation of cannabinoid-induced cell death, and of cell proliferation upon genetic or pharmacologic ma- nipulation of the endocannabinoid system—are therefore needed to clarify the contextual determinants that result in the system acting as either a guardian or an inducer of tumourigenesis or tumour progression. The present review summarizes such observations and provides an integrated view of the molecular mechanisms responsible for cannabinoid antitumour activity. It also discusses the experimental evidence supporting the existence of
mechanisms of resistance to the cell death–promoting ac- tions of thc in certain types of cancer cells, the strategies that could possibly be used to overcome such resistance, and the preclinical data supporting the potential useful- ness of the combined administration of cannabinoids and other drugs in anticancer therapies.
PRECLINICAL ANTITUMOUR ACTIVITY
Since the late 1990s, a large body of evidence has accu- mulated demonstrating that various cannabinoids exert antitumour effects in a wide variety of experimental mod- els of cancer, ranging from cancer cell lines in culture to genetically-engineered mice (reviewed by Velasco et al.17). Multiple cannabinoids have shown this activity, including thc; the endocannabinoids 2-arachidonoylglycerol and anandamide; and various synthetic cannabinoid receptor agonists that have either comparable af nity for the cb1 and cb2 receptors (for example, WIN 55,212-2 or HU-210), a higher af nity for cb1 (for example, methanandamide), or a higher af nity for cb2 (for example, JWH-133). Those ndings strongly support that, aside from the role played by the endogenous cannabinoid system in cancer, phar- macologic stimulation of cb receptors is, in most cases, antitumourigenic. Nonetheless, a few reports have pro- posed a tumour-promoting effect of cannabinoids18–21. Those apparently con icting observations are discussed in the next subsection.
Mechanisms of Antitumour Effects
Cannabinoids impair tumour progression at various levels. Their most prevalent effect is the induction of cancer cell death by apoptosis and the inhibition of cancer cell prolif- eration. At least one of those actions has been demonstrated in almost all cancer cell types tested17. In addition, in vivo experiments have shown that cannabinoids impair tumour angiogenesis and block invasion and metastasis.
Induction of Cancer Cell Death
A signi cant amount of the research conducted so far on the mechanism of cannabinoid antitumour activity has focussed on glioma cells. Initial studies showed that thc and other cannabinoids induce the apoptotic death of glioma cells by cb1- and cb2-dependent stimulation of the de novo synthesis of the pro-apoptotic sphingolipid ceramide23,32–34. Further studies based on the analysis of the gene expression pro le of thc-sensitive and -resistant glioma cells yielded further insight into the speci c sig- nalling events downstream of ceramide that are activated in cancer cells by cannabinoids35. Thus, it was found that treatment with thc results in enhanced expression of the stress-regulated protein p8 (nupr1), a transcriptional regulator that has been implicated in the control of tu- mourigenesis and tumour progression36, together with several of its downstream targets, such as the endoplasmic reticulum (er) stress–related transcription factors atf4 and chop, and the pseudokinase tribbles homologue 3 (trib3)35. This thc-triggered stimulation of the p8-regulated pathway (Figure 1) enhances the inhibitory interaction of trib3 with a pro-survival kinase, akt37,38, which leads to inhibition of the mammalian target of rapamycin complex 1 (mtorc1)
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Cannabinoid-induced apoptosis relies on the stimulation of endoplasmic reticular (ER) stress and autophagy. Here, the mecha- nism of cannabinoid-induced apoptosis in glioma, pancreatic, and hepatocellular carcinoma cells is depicted. This signalling route could constitute the main mechanism of cannabinoid-induced cell death, with some variations inherent to different types of cancer cells. CB1 = can- nabinoid CB1 receptor; CB2 = cannabinoid CB2 receptor; SPT = serine palmitoyltransferase; elF2α = eukaryotic translation initiation factor 2α; P inscribed in a circle = protein phosphorylation upon treatment with Δ9-tetrahydrocannabinol (THC); ATF-4 = activating transcription factor 4; CHOP = C/EBP homologous protein; AKT = protein kinase B; TRIB3 = tribbles-homologue 3; mTORC2 = mammalian target of rapamycin complex 2; CDKs = cyclin-dependent kinases; pRb = retinoblastoma protein; CaCMKKβ = calcium/calmodulin–dependent protein kinase kinase 2β; AMPK = AMP-activated protein kinase.
ANTICANCER MECHANISMS OF CANNABINOIDS, Velasco et al.
FIGURE 1
The importance of this pathway is highlighted by the ability of various chemical and genetic manipulations to block cannabinoid-induced cell death. In hepatocellular carcinoma cells, the cannabinoid-evoked and er stress– dependent activation of calcium/calmodulin-dependent protein kinase kinase 2β and amp-activated protein kinase leads, together with the p8–trib3 pathway, to autophagy and apoptosis43. The cannabinoid-evoked inhibition of akt was able to promote cycle arrest in breast cancer and melanoma cells, as well as apoptosis through additional mechanisms, including decreased phosphorylation of the pro-apoptotic protein bcl2-associated agonist of cell death45 and activation of the cyclin-dependent kinase inhibitory proteins p21 and p2724,46,47, leading to the sub- sequent decreased phosphorylation of the retinoblastoma protein, which would thus arrest the cell cycle.
The direct participation of the p8/trib3–mediated au- tophagy pathway in the antitumour action of cannabinoids has been clearly demonstrated in glioma cells, pancreatic and hepatic cancer cells, and melanoma cells35,37,43,44,48,49. At least part of that signalling route has also been found to be upregulated with cannabinoid treatment in other types of cancer cells, an observation which suggests that—with some variations—this pathway could be a general mecha- nism by which activation of cb1 and cb2 receptors promotes cancer cell death.
Additional mechanisms might nonetheless cooperate with the p8/trib3–mediated autophagy pathway to evoke cancer cell death (Figure 1). For example, in hepatocel- lular carcinoma cells, thc and the cb2 receptor agonist JWH-015 can trigger an er stress–dependent activation of amp-activated protein kinase that cooperates with the trib3-mediated inhibition of the akt–mtorc1 axis in the stimulation of autophagy-mediated cell death44. In mela- noma46, breast carcinoma24,50, and prostate carcinoma51 cells, cannabinoids can induce cell-cycle arrest in concert with apoptosis24,46,51. Notably, cannabinoid antiprolifera- tive action—at least in melanoma46 and breast cancer24 cells—also relies on akt inhibition.
Likewise, the effect of cannabinoids in hormone- dependent tumours might rely, at least in part, on the ability to interfere with the activation of growth factor receptors12,16. Some of those and other mechanisms52 might participate, together with the autophagy-mediated cell death pathway, in the cytotoxic action of cannabinoids in various types of cancer cells. However, further investiga- tion is required to clarify the issue.
Research conducted during the last few years has shed light on the intracellular signalling mechanisms underly- ing cannabinoid anticancer action. However, a number of important observations—in particular, those related to the role played by cannabinoid receptors in the trigger- ing of the signals—remain to be clari ed. For example, in contrast to the death-promoting action of cannabinoids on cancer cells, the viability of normal (non-transformed) cells is unaffected or, under certain conditions, even en- hanced by cannabinoid challenge33–35,37,53. For example, thc treatment of astrocytes (a cell type that expresses functional cb1 receptors) does not trigger the activation of er stress, upregulation of the p8 pathway, inhibition of the akt–mtorc1 axis, or stimulation of autophagy and
and the subsequent stimulation of autophagy-mediated cell death37.
Autophagy is an essential cellular process participat- ing in a number of physiologic functions within the cell39,40. During autophagy, organelles and other cytoplasmic com- ponents are engulfed within double-membrane vesicles called “autophagosomes.” The maturation of those vesicles involves their fusion with lysosomes, which leads in turn to the degradation of the autophagosome components by lysosomal enzymes40. Autophagy is primarily a cytopro- tective mechanism, although its activation can also lead to cell death40–42.
Cannabinoids induce autophagy in various types of cancer cells in culture, and pharmacologic or genetic in- hibition of autophagy prevents cannabinoid antitumour action in various animal models of cancer (Figure 1), thus demonstrating that autophagy is important for cannabi- noid antineoplastic activity37,43,44. Moreover, autophagy blockade prevents cannabinoid-induced apoptosis and cell death, whereas apoptosis blockade prevents cannabinoid- induced cell death, but not autophagy37,43,44. Those obser- vations indicate that autophagy is upstream of apoptosis in the mechanism of cannabinoid-induced cell death (Figure 1).
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apoptosis, even when concentrations of thc higher than those that promote glioma cell death are used35,37. Similar results were obtained for primary embryonic broblasts35,41 and other types of non-transformed cells expressing func- tional cannabinoid receptors in comparison with their transformed counterparts24,46,54,55. Thus, stimulation of cannabinoid receptors seems to be coupled with the acti- vation of different signalling mechanisms in transformed and non-transformed cells. The precise molecular reasons for this variation in behaviour remain as an important open question in the cannabinoid eld. Another intrigu- ing observation is that, in some types of cancer cells (such as glioma cells), pharmacologic blockade of either cb1 or cb2 prevents cannabinoid-induced cell death with similar ef cacy33,56, and yet in other types of cancer cells (pancre- atic48, breast24, or hepatic43 carcinoma cells, for example), antagonists of cb2 but not of cb1 inhibit cannabinoid an- titumour actions. The reason that cannabinoids produce their antitumour actions through one or the other of these receptor types depending on the type of cancer cell has yet to be established.
Some cannabinoid receptor agonists promote cancer cell death more ef ciently than other agonists that exhibit similar or even higher af nity for the cb1 or cb2 receptors. For example, thc promotes cancer cell death in a cb1- or cb2-dependent manner (or both) at lower concentrations than does the synthetic cannabinoid receptor agonist WIN 55,212-2, although the latter agent shows signi cantly higher af nity for cb1 and cb2 in binding assays6.
Further work aimed at investigating, for example, cb receptor homo- or hetero-oligomerization in response to various cannabinoid agonists, their associations with spe- ci c domains in the plasma membrane such as lipid rafts, changes in the subcellular location of cb receptors, and the selective coupling to various G proteins and other signalling proteins, will be essential to answer the foregoing questions and to precisely de ne the role played by each cannabinoid receptor type as an anticancer signalling platform.
Notably, cannabidiol (cbd), a phytocannabinoid with a low af nity for cannabinoid receptors15, and other mar- ijuana-derived cannabinoids57 have also been proposed to promote the apoptotic death of cancer cells acting in- dependently of the cb1 and cb2 receptors. The mechanism by which cbd produces this effect has not as yet been com- pletely clari ed, but it seems to rely—at least in part—on its ability to enhance the production of reactive oxygen species in cancer cells58,59. It has also been proposed that cbd might activate trpv2 receptors to promote glioma cell death60.
Inhibition of Angiogenesis, Invasion, and Metastasis
In cancer cells, cannabinoids block the activation of the vascular endothelial growth factor (vegf) pathway, an inducer of angiogenesis. Speci cally, various elements of the cascade, such as the main ligand (vegf) and the active forms of its main receptors (vegfr1 and vegfr2), are downregulated with cannabinoid treatment of skin carcinomas54, gliomas32,61, and thyroid carcinomas62. In vascular endothelial cells, cannabinoid receptor activation inhibits proliferation and migration, and induces apop- tosis61,63. Those and perhaps other cannabinoid-evoked actions result in a normalized tumour vasculature—that
is, smaller and fewer vessels that are more differentiated and less leaky.
Likewise, cb1 or cb2 receptor agonists (or both) re- duce the formation of distant tumour masses in animal models of both induced and spontaneous metastasis, and inhibit adhesion, migration, and invasiveness of glioma64, breast65,66, lung67,68, and cervical68 cancer cells in culture. Those effects depend, at least in part, on the modulation of extracellular proteases (such as matrix metalloprotein- ase 2)64 and their inhibitors (such as tissue inhibitor of matrix metalloproteinases 1)68.
Notably, pharmacologic inhibition of ceramide bio- synthesis abrogates the antitumour and antiangiogenic effect of cb1 or cb2 receptor agonists (or both) in glioma xenografts, and decreases vegf production by glioma cells in vitro and in vivo32. Likewise, inhibition of matrix metalloproteinase 2 expression and glioma cell invasion is prevented by blocking ceramide biosynthesis and by knocking down p8 expression64. Although further research is still necessary to precisely de ne the molecular mecha- nisms responsible for those actions of cannabinoids, the observations indicate that the ceramide/p8–regulated pathway plays a general role in the antitumour activity of cannabinoids targeting cb1 and cb2.
It is worth noting that cbd, by acting independently of the cb1 and cb2 receptors, produces a remarkable anti- tumour effect—including reduction of invasiveness and metastasis—in various animal models of cancer. This effect of cbd seems to rely, at least in part, on the downregulation of the helix-loop-helix transcription factor inhibitor of dna binding 169,70.
Regulation of Antitumour Immunity
Notably, stimulation of cannabinoid receptors can lead to important changes in the processes that regulate anti- tumour immunity. Thus, for example, treatment of mice with thc triggers a shift (from Th1 to Th2) of cytokine pro le20,71–73 and induces mobilization of myeloid-derived suppressor cells74, two events that play a critical role in the suppression of antitumour immunity. In agreement with that notion, stimulation of cb2 has been proposed in some reports to enhance tumourigenesis by interfering with tumour surveillance by the immune system20,21.
By contrast, cannabinoids can also enhance immune system–mediated tumour surveillance in some contexts: the antitumour action of WIN 55,212-2 (a mixed cb1 or cb2 agonist) or JWH-133 (a cb2-selective agonist) was more pronounced in melanoma xenografts generated in immunocompetent mice than in those generated in im- munode cient mice46, a nding which also indicates that, at least in this model, stimulation of cb2 inhibits tumour growth primarily through direct effects on cancer cells rather than necessarily by interfering with the normal antitumour function of the immune system. In line with that idea, treatment of immunocompetent rats with very high doses of thc (50 mg/kg daily 5 times per week) for 2 years lowered the incidence of several types of tumours and enhanced the overall survival of the animals55. Those observations might be related to the ability of thc to reduce in ammation75,76, an effect that might prevent certain types of cancer76,77.
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For cannabinoid use to be clinically successful, anti- tumour effects will have to overcome immunosuppressive (potentially tumour-promoting) effects. Additional studies should clarify the issue. For example, it could be conceiv- able to study the effect of cannabinoid administration on the generation and progression of tumours with varying sensitivity to cannabinoids and generated in immunocom- petent or immunode cient mice in which the expression of cb1 or cb2 receptors (or both) in cells from the immune system has been genetically manipulated.
RESISTANCE MECHANISMS
Numerous studies have contributed to an appreciation of the heterogeneity of cancer, whereby each subtype of cancer—and even each individual tumour—exhibits a series of molecular characteristics that determines its behaviour and, in particular, its responsiveness to various anticancer drugs. In agreement with that line of reason- ing, a recent report investigated the molecular features associated with the resistance of a collection of human glioma cell lines and primary cultures to cannabinoid antitumour action56. The study showed that, although the apoptotic effect of thc on glioma cells relied on the stimulation of cannabinoid receptors and activation of the p8-mediated autophagy pathway, the differences in the sensitivity to thc-induced cell death correlated with enhanced expression of a particular set of genes in the thc-resistant glioma cells rather than with the presence of different expression levels of cb1 or cb2 receptors56. Interestingly, upregulation of one of those genes, midkine (MDK ), which encodes a growth factor that was previously associated with increased malignancy and resistance to anticancer therapies in several types of tumours77,78, cor- relates with lower overall survival in patients with glioblas- toma56. Moreover, mdk plays a direct role in the resistance to thc action through stimulation of anaplastic lymphoma kinase (alk79). Thus, the stimulation of alk by mdk inhibits the thc-evoked autophagy-mediated cell-death pathway. Further research should clarify whether that mechanism could also be responsible for the resistance to other thera- pies of cancer cells expressing high levels of mdk. Interest- ingly, in vivo silencing of MDK or pharmacologic inhibition of alk in a mouse xenograft model abolishes the resistance to thc treatment of established tumours derived from cannabinoid-resistant glioma cells56.
Taken together, the foregoing ndings support the idea that stimulation of the mdk–alk axis promotes resistance to thc antitumour action in gliomas and could help to set a foundation for the potential clinical use of thc in com- bination with inhibitors of the mdk–alk axis (Figure 2). Glioblastoma is highly resistant to current anticancer therapies80–82. Speci cally, resistance of glioma cells to cannabinoid-induced cell death relies, at least in part, on enhanced expression of mdk and the subsequent activation of alk56. Likewise, enhanced expression of the heparin- bound epidermal growth factor receptor (egfr) ligand amphiregulin can promote resistance to thc antitumour action by stimulation of extracellular signal-regulated ki- nase (erk)83. The combination of thc with pharmacologic inhibitors of alk (or genetic inhibition of MDK) enhances
cannabinoid action in resistant tumours, which provides a rationale for the design of targeted therapies capable of increasing cannabinoid antineoplastic activity56. Combi- nations of cannabinoids with classical chemotherapeutic drugs such as the alkylating agent temozolomide (the benchmark agent for the management of glioblastoma80,84) have been shown to produce a strong anticancer action in animal models85. Combining cannabinoids and temozolo- mide is thus a very attractive possibility for clinical studies aimed at investigating cannabinoid antitumour effects in glioblastoma. Other potentially interesting strategies to enhance cannabinoid anticancer action (still requiring additional experimental support from data obtained using preclinical models) could be to combine cannabinoids with er stress or autophagy inducers (or both) or with inhibitors of the mtorc1 axis.
In line with that idea, alk inhibitors have started to be used in clinical trials for the management of non- small-cell lung cancer and other types of tumours86,87. Future research should clarify whether this mechanism of resistance to cannabinoid action operates in other types of tumours. In agreement with that possibility, MDK silencing enhanced the sensitivity of cannabinoid-resistant pancre- atic cancer cells to thc-induced cell death56.
The release by cancer cells of other growth factors has also been implicated in the mechanism of resistance to cannabinoid antitumour action. Thus, increased expres- sion of amphiregulin is associated with enhanced resis- tance to thc antitumour action in glioma xenografts83. Notably illustrating how the dose of cannabinoids could be crucial for optimal therapeutic effect, low (submicro- molar) concentrations of thc or other synthetic cannabi- noid agonists enhance the proliferation of several cancer cell lines in vitro. That effect relies on activation of the protease adam17, the shedding of heparin-bound egfr li- gands including amphiregulin and the subsequent stim- ulation of the erk and akt pathways19. In line with that idea, a recent report showed that treatment with the syn- thetic cannabinoid CP 55,940 increases the proliferation of murine glioma cells engineered to express cb1 or cb2 receptors only when those receptors are coupled to akt activation18. Although a pro-tumourigenic effect has not been observed for the growth of tumour xenografts gener- ated with glioma cells and treated with low doses of thc85, increased expression of amphiregulin promotes resistance to thc antitumour action through a mechanism that in- volves the egfr-dependent stimulation of erk and the subsequent inhibition of p8 and trb3 expression. Likewise, pharmacologic inhibition of egfr, erk83, or akt enhances the cell-death-promoting action of thc in glioma cultures (unpublished observations by the authors), which suggests that targeting egfr and the akt and erk pathways could enhance the antitumour effect of cannabinoids.
CANNABINOID-BASED COMBINATION THERAPIES
The use of combinational anticancer therapies has a number of theoretical advantages over single-agent strat- egies, because they allow for the simultaneous targeting of tumour growth, progression, and spread at various
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levels. In line with that idea, recent observations suggest that the combined administration of cannabinoids with other anticancer drugs acts synergistically to reduce tu- mour growth. For example, the administration of thc and temozolomide exerts strong antitumour action in glioma xenografts, an effect that is also evident in temozolomide- resistant tumours85. A similar effect was observed when thc and cbd were combined with radiotherapy in animal models of glioma. Interestingly, no toxicity was observed in mice treated with combinations of thc and temozolo- mide85. Because most patients with glioblastoma undergo temozolomide treatment, the foregoing ndings indicate that the combined administration of temozolomide and cannabinoids could be therapeutically exploited for the management of glioblastoma (Figure 2) and perhaps other tumour types such as melanoma44.
Likewise, another study recently showed that the combined administration of gemcitabine (the benchmark agent for the treatment of pancreatic cancer) and various cannabinoid agonists synergistically reduced the viability of pancreatic cancer cells88. Other reports indicated that anandamide and HU-210 might also enhance the antican- cer activity of paclitaxel89 and 5- uorouracil90 respectively.
An additional approach has been to combine thc with cbd, a phytocannabinoid that reduces (although to a lower extent than thc) the growth of several types of tumour xe- nografts through a still poorly-de ned mechanism59,91,92. Combined administration of thc and cbd enhances the anticancer activity of thc and reduces the dose of thc needed to induce its tumour growth-inhibiting activity85,93. Moreover, the combination of thc and cbd together with temozolomide produces a striking reduction in the growth of glioma xenografts even when low doses of thc are used85. Likewise, the combination of thc, cbd, and radiotherapy also produced clear anticancer activity in an orthotopic model of glioma94. Notably, cbd was also shown to alleviate some of the undesired effects of thc administration such as convulsions, discoordination, and psychotic events, thus improving the tolerability of cannabis-based medicines15. As mentioned earlier, C. sativa produces approximately 108 dif- ferent cannabinoids, and apart from cbd, some of the other cannabinoids present in marijuana might attenuate the psychoactive side effects of thc or even produce other thera- peutic bene ts15. Thus, we think that clinical studies aimed at analyzing the ef cacy of cannabinoids as antitumour agents should be based on the use both of pure substances, such as thc and cbd, and of cannabis extracts containing controlled amounts of thc, cbd, and other cannabinoids.
CLINICAL ANTITUMOUR EFFECTS OF CANNABINOIDS
The clinical approval of cannabinoids is largely restricted to palliative uses in various diseases, but since the emer- gence of promising preclinical data, the antitumour effects of cannabinoids are beginning to be clinically assessed.
In a pilot phase i clinical study, 9 patients with actively- growing recurrent glioblastoma for whom standard ther- apy had previously failed underwent intracranial thc administration11. Under those conditions, cannabinoid delivery was safe and could be achieved without signi cant
FIGURE 2
unwanted effects. In addition, although no statistically signi cant conclusions can be extracted from a cohort of 9 patients, the results obtained in the study suggest that some patients responded—at least partially—to thc treat- ment in terms of a decreased tumour growth rate as eval- uated by magnetic resonance imaging11. Importantly, analyses of samples obtained from 2 study patients before and after thc administration indicated that the molecular mechanism of cannabinoid antitumour action—namely, p8 and trib3 upregulation35,37, mtorc1 inhibition37, stim- ulation of autophagy and apoptosis11,35,37, inhibition of cell proliferation11, decreased vegf signalling32, and matrix metalloproteinase 2 downregulation64 (delineated here earlier)—also operates in vivo.
Those ndings were encouraging and reinforced in- terest in the potential use of cannabinoids in cancer ther- apies. However, they also highlighted the need for further research aimed at optimizing the use of cannabinoids in terms of patient selection, combination with other anti- cancer agents, and use of other routes of administration.
Administration of endocannabinoids or inhibitors of endocannabinoid-degrading enzymes has been shown to reduce the growth of various tumour xenograft types95,96 and could therefore be a reasonable strategy for targeting cannabinoid receptors for anticancer purposes. However, as discussed here earlier, the role of the endocannabi- noid system, including the endocannabinoid-degrading enzymes, in the control of tumour generation and pro- gression is not well understood. Because enhancing endocannabinoid tone only has mild antitumour effects
Possible strategies for optimizing cannabinoid-based therapies against gliomas. GF = growth factors; Abs = antibodies; MDK = growth factor midkine; VEGF = vascular endothelial growth factor; RTK = receptor tyrosine kinase; ALK = anaplastic lymphoma receptor tyrosine kinase; EGFR = epidermal growth factor receptor; CB1 = cannabinoid CB1 receptor; CB2 = cannabinoid CB2 receptor; AREG = amphiregulin; ER stress = endoplasmic reticular stress; ERK = extracellular signal-regulated kinase; TMZ = temozolomide; TRIB3 = tribbles-homologue 3; AKT = protein kinase B; mTORC1 = mammalian target of rapamycin complex 1.
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in mice and because no inhibitor of endocannabinoid degradation has yet been approved for use in humans, clinical studies aimed at analyzing the ef cacy of can- nabinoids as antitumour agents should be based on the use of plant-derived or synthetic agonists of cannabinoid receptors rather than on endocannabinoids or inhibitors of endocannabinoid degradation.
The long-known therapeutic properties of C. sativa— including amelioration of symptoms associated with cancer and its chemotherapy—have led to the authorization of the medical use of the plant and its extracts in several countries. As already mentioned, C. sativa produces approximately 108 different cannabinoids, including thc and cbd. Some of the other cannabinoids present in marijuana might contribute to the attenuation of thc’s psychoactive side effects or even to the production of other therapeutic bene ts15. However, pure drugs are more prone to standardization than complex mo- lecular cocktails. Thus, it would be ideal for studies aiming to investigate the anticancer actions of cannabinoids in patients to be performed comparatively with both pure substances and cannabis extracts containing controlled amounts of thc, cbd, and other cannabinoids.
The most widely used route of administration for recreational and self-medicating marijuana is smoking. Although thc and other phytocannabinoids are rapidly absorbed by inhalation, smoking is an unattractive clini- cal option. Preclinical work in animal models has typically used peri-tumoural administration of cannabinoids. Like- wise, in the only clinical trial in which a cannabinoid was assayed as an antitumour agent, thc was administered locally (intracranial delivery to patients with glioblastoma multiforme)11. Nevertheless, this route of administra- tion has many obvious limitations. Currently available cannabis-based medicines are administered as capsules or using an oromucosal spray15. Preclinical animal models have yielded data indicating that systemic (oral or intra- peritoneal) administration of cannabinoids effectively decreases tumour growth (GV, CS, and MG. Unpublished observations), and so it seems reasonable that future clinical studies with the goal of determining the ef cacy of cannabinoids as antitumour agents use oral or oromucosal routes of administration.
Two currently ongoing clinical trials could shed some light on these issues. One of the studies is a phase i/ii trial aimed at evaluating the combined effect of Sativex (an oromucosal cannabis extract whose main active compo- nents are thc and cbd in a c. 1:1 ratio) and temozolomide in patients with recurrent glioblastoma multiforme (https:// clinicaltrials.gov/ct2/show/NCT01812603 and https:// clinicaltrials.gov/ct2/show/NCT01812616). The other is a phase ii trial aimed at evaluating the effect of cbd as single treatment in patients with solid tumours (https://clinical trials.gov/ct2/show/NCT02255292). Hopefully, in the near future, new clinical trials will start, helping to determine whether cannabinoids can be used, for other than their palliative effects, in the treatment of cancer patients
CONCLUSIONS AND FUTURE DIRECTIONS
It is widely believed that strategies aimed at reducing mortal- ity from cancer should consist of targeted therapies capable
of providing the most ef cacious and selective treatment for each individual tumour and patient. Thus, the major focus of anticancer drug development has progressively moved from nonspeci c chemotherapies to molecularly-targeted inhibitors. However, despite the huge amount of preclini- cal literature on how these rationally designed compounds work, their use in clinical practice is still limited.
How do cannabinoid-based medicines t into this ongoing scenario? Consider glioma, the type of cancer in which the most detailed cannabinoid research has been conducted to date. As discussed here, engagement of a molecular target (the cb receptors) by a family of selective drugs (thc and other cannabinoid agonists) inhibits tu- mour growth in animal models through a well-established mechanism of action that also seems to operate in human patients. Moreover, cannabinoids potentiate the antitu- mour ef cacy of temozolomide and alk inhibitors in mice harbouring gliomas. Those ndings provide preclinical proof-of-concept that “cannabinoid sensitizers” could improve the clinical ef cacy of classical cytotoxic drugs in glioblastoma (Figure 2) and perhaps other highly ma- lignant tumours such as pancreatic cancer, melanoma, and hepatocellular carcinoma. However, further research is required to de ne the precise molecular cross-talk be- tween cannabinoids and chemotherapeutic drugs and to optimize the pharmacology of preclinical cannabinoid- based combination therapies.
With respect to patient strati cation, the particular individuals that are potentially responsive to cannabinoid administration should be unequivocally determined. To that end, high-throughput approaches should be imple- mented to nd cannabinoid therapy–associated biomark- ers in tumour biopsies or, ideally, in easily acquired uids containing circulating cancer cells or enhanced levels of resistance factors that might have been released by cancer cells. Such biomarkers would conceivably relate to can- nabinoid pharmacodynamics—namely, expression and activity of cannabinoid receptors and their downstream cell-death-inducing effectors. The approach would be analogous to the biochemical evaluation of estrogen and ErbB2 receptors, which respectively predict bene t from endocrine therapies and trastuzumab in breast cancer. Predictive markers to de ne the sensitivity of a particular tumour to cannabinoid-based therapies could also include the status of growth factors, such as mdk in gliomas, and their receptors and signalling partners.
To summarize, cannabinoids induce tumour cell death and inhibit tumour angiogenesis and invasion in animal models of cancer, and there are indications that they act similarly in patients with glioblastoma. Given that can- nabinoids show an acceptable safety pro le, clinical trials testing them as single drugs or, ideally, in combination therapies in glioblastoma and other types of cancer are both warranted and urgently needed.
ACKNOWLEDGMENTS
Research by the authors and colleagues in their groups is sup- ported by grants from the Spanish Ministry of Economy and Competitiveness and the Fondo Europeo de Desarrollo Regional (PI12/02248, PI15/0339, FR2009-0052, and IT2009-0053 to GV, and PI11/00295 to CS), Comunidad de Madrid (S2011/BMD-2308
ANTICANCER MECHANISMS OF CANNABINOIDS, Velasco et al.
Current Oncology, Vol. 23, Supp. 2, March 2016 © 2016 Multimed Inc.
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ANTICANCER MECHANISMS OF CANNABINOIDS, Velasco et al. to MG), GW Pharmaceuticals (to GV and CS), Fundación Mutua
Madrileña (AP101042012 to GV), and Fundació La Marató de TV3 (20134031 to GV).
CONFLICT OF INTEREST DISCLOSURES
We have read and understood Current Oncology’s policy on dis- closing con icts of interest, and we declare the following inter- ests: GW Pharmaceuticals and Cellmid fund part of the research conducted by our laboratory. Likewise, a portion of the data obtained by the authors concerning the antitumoural action of cannabinoids is included in three patent applications presented by GW Pharmaceuticals.
AUTHOR AFFILIATIONS
*Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, and Instituto Universita- rio de Investigación Neuroquímica, Madrid, Spain; †Centro de Investigación Biomédica en Red sobre Enfermedades Neurode- generativas, Madrid, Spain; ‡Instituto de Investigación Sanitaria del Hospital Clínico San Carlos, Madrid, Spain; §Instituto de Investigación Hospital 12 de Octubre, Madrid, Spain; ||Instituto Ramón y Cajal de Investigación Sanitaria, Madrid, Spain.
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