Molecular Targets of Minor Cannabinoids in Breast Cancer: In Silico and In Vitro Studies

Abstract

1. Introduction

Despite the continuous advances in drug development, breast cancer incidence keeps rising, having the main responsibility for all cancer-related death among women worldwide [1,2]. Due to their great heterogeneity, breast tumors are grouped in different subtypes, with most of them being classified as estrogen receptor-positive (ER⁺) [3,4,5]. This subtype overexpresses estrogen receptor alpha (ERα) and progesterone receptor (PR). Moreover, in these tumors, the enzyme aromatase is overexpressed, resulting in high rates of local estrogen production [4,6,7]. The elucidation of these targets was pivotal for the development of endocrine therapies, which prevent estrogen production or signaling, such as aromatase inhibitors (AIs), including Anastrozole (Ana), Letrozole (Let), and Exemestane (Exe), as well as anti-estrogens, such as Tamoxifen and Fulvestrant (Figure 1) [8]. In fact, these drugs are part of the therapeutic strategy applied as first-line therapy, both in pre- and post-menopausal status [8,9,10,11,12]. Unfortunately, and despite their clinical success, these compounds are not only associated with adverse side effects, but also with the development of endocrine resistance, which compromises their effectiveness [13,14]. In view of that, alternative therapeutic approaches have been developed to improve treatment, including the introduction of new targeted therapies with novel drugs such as CDK4/6, mTOR, and PI3K inhibitors that are recommended in combination with endocrine therapy [11,12,13,14,15,16,17]. Indeed, the elucidation of the role of additional targets in the development of ER⁺ breast cancer represents a new hope for its treatment. One of the targets that has been considered is the androgen receptor (AR), which is expressed in 70–90% of ER⁺ breast tumors [13,18,19]. This receptor seems to cooperate with ERα and displays different functions depending on the hormonal and resistance status. In ER⁺ breast cancer cases, AR antagonizes ERα signaling, contributing to the decrease in cell proliferation and tumor growth [20]. On the other hand, in cases where resistance to endocrine therapy develops, and thus ERα activity is modified, AR switches its function from tumor suppressor to oncogenic in order to promote cell growth and survival. This emphasizes the existence of a hormone-dependent crosstalk between ERα and AR [21,22,23,24].

Cannabinoids have demonstrated promising therapeutic actions in different pathological conditions, including epilepsy, asthma, sleep disorders, depression, and inflammation, and in the relief of chemotherapy-associated side effects [27,28,29]. Notably, various studies have attributed anti-proliferative, anti-angiogenic, and anti-invasive actions to cannabinoids in various cancer types [28,30,31,32,33,34,35,36,37]. In fact, the in vitro and in vivo effects of the two best known cannabinoids, Δ⁹-tetrahydrocannabinol (THC) and cannabidiol (CBD), were already explored in different cancer types, including breast cancers. Usually, the cannabinoids exert anti-tumor effects through the inhibition of cell growth, neovascularization, migration, adhesion, invasion, and metastasis, and also by promoting cell cycle arrest, autophagy, and apoptosis [33,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57]. To date, the studies conducted on cannabinoids in breast cancer were mainly performed in triple-negative and HER2⁺ tumors [33,58,59,60]. Nevertheless, there is evidence that molecular pathways between cannabinoid receptors and estrogens or androgens may overlap, which may impact ER⁺ breast cancer [60,61]. In 1997, Ruh et al. demonstrated that THC and CBD fail to act as ER agonists in MCF-7 cells [62]. Years later, it was demonstrated that THC impaired proliferation of MCF-7 cells by an ER- and AR-independent mechanism [63], and disrupted estrogen-signaling by up-regulating ERβ [55]. More recently, our group has clarified the anti-cancer role of CBD and THC, and of the endocannabinoid anandamide (AEA) in MCF-7aro cells, unveiling their mechanism of action and their ability to modulate key targets, such as aromatase, ERα, and ERβ [42,64,65]. Besides this, we have verified that CBD and AEA fit well at the aromatase binding site, inhibiting its activity by 78.6% and 61.0%, respectively, at 2 μM, in human placental microsomes [64,65], whereas THC inhibited 25.6% [65]. Moreover, we recently demonstrated that CBD acts as an ER and AR antagonist, with inverse agonist properties at both receptors, depending on hormonal influence. Furthermore, this cannabinoid is able to improve AIs’ anti-proliferative effects in ER⁺ breast cancer cells, with the combination of CBD with Exe having potential clinical value as an adjuvant therapy [44]. However, Cannabis contains several other phytocannabinoids. In fact, more than 500 different compounds were isolated from the plant and around 140 of them are phytocannabinoids. Regarding Cannabis sativa, 129 phytocannabinoids have been described whose therapeutic potential has not been fully understood. They are generally grouped in 11 different classes: the Δ⁹-THC type, Δ⁸-THC type, CBD type, cannabigerol (CBG) type, cannabinodiol (CBND) type, cannabielsoin (CBE) type, cannabicyclol (CBL) type, cannabichromene (CBC) type, cannabinol (CBN) type, cannabitriol (CBT) type, and miscellaneous type [27,66,67,68,69]. All the cannabinoids included in the different classes share a similar C21 terpenophenolic backbone and their biosynthesis is mostly derived from the geranyl diphosphate (GPP) prenylation of either divarinolic acid or olivetolic acid, a reaction catalyzed by GPP olivetolate geranyltransferase. As a result, cannabigerovarinic acid (CBGVA) and cannabigerolic acid (CBGA) are produced, respectively. Through enzymatic and non-enzymatic transformations, CBGVA and CBGA originate the phytocannabinoids that constitute the five main classes described so far (Figure 2) [70,71]. The pharmacological interest about the minor phytocannabinoids has been growing and the anti-tumor properties of CBG, CBN, cannabidiolic acid (CBDA), cannabidiol-C4 (CBDB), and cannabichromenic acid (CBCA) were already addressed in diverse types of cancers, including breast [28,45,72,73,74,75,76,77,78,79,80,81]. So far, only the minor phytocannabinoids CBG and CBN have been studied in breast cancer models [75,76]. Taken together, we aimed to evaluate, in silico and in vitro, the ability of minor phytocannabinoids to interact with and modulate important targets in developing ER⁺ breast cancer, namely aromatase, ERα, and AR, to expand our knowledge on these compounds and substantiate the development of new therapeutic solutions. Moreover, we intend to characterize the potential multi-target actions of cannabinoids in this disease.

2. Results

3. Discussion

The use of endocrine therapies, such as AIs and anti-estrogens, for ER⁺ breast cancer treatment has been facing concerns and limitations mainly due to endocrine resistance development, which has led to a search for their combination with other innovative therapies. However, these therapies also cause resistance [13,94,95,96], which is the reason why the discovery and development of novel therapeutic approaches are necessary. Various studies on the anti-tumor effects of cannabinoids in different cancer forms have been performed, showing promising results. Indeed, our group has demonstrated that THC and CBD decrease aromatase and ERα protein levels in ER⁺ breast cancer cells and that CBD can also inhibit aromatase [42] and presents ER and AR antagonistic, as well as inverse agonist, properties, in both receptors [44]. Other studies reported that THC presents anti-proliferative effects in ER⁺ breast cancer cells independent of ER and AR [63], although having the ability to impair ERα signaling by up-regulating ERβ [55]. Moreover, it was already described that THC and CBD exert no ER agonistic activity [62]. Considering all this, the ability of the known 129 phytocannabinoids to modulate key targets associated with ER⁺ breast cancer progression was investigated in this study.

Due to its fundamental role in local estrogen production and tumor development, aromatase was the first target studied. Our in silico studies predicted that 45 out of the 129 phytocannabinoids may act as AIs. Besides the binding affinity score, one of the reasons for their selection was the interactions that they, in theory, manage to establish with this enzyme, such as a hydrogen bond with the Asp309 residue, considered fundamental for the aromatization reaction [84,85]. In fact, the AIs used in the clinic do bind to this key residue, thereby presenting high anti-aromatase activity [83,97,98]. ERα, a key target that through estrogen binding promotes the signal transduction necessary for tumor growth and survival, was also explored. In this case, 85 cannabinoids out of the 129 showed important binding features that led us to consider them as potential ERα antagonists. One of the most important features taken into consideration was their ability to be localized in the vicinity of helix 12, a flexible helix of ERα that, upon antagonist binding, prevents the binding of coactivators, compromising receptor activation [99]. On the other hand, their possible proximity and establishment of hydrogen bonds with Asp351, a residue close to helix 12 and to which antagonists typically bind, were also taken into account [6]. Considering these results, it is possible to suggest that, theoretically, cannabinoids have a higher potential to bind and act as ERα antagonists than as AIs. Furthermore, 36 compounds out of the 45 potential AIs and of the 85 potential ER antagonists were simultaneously considered as both promising AIs as well as ERα antagonists, suggesting that they may act as multi-target compounds in ER⁺ breast cancer. In fact, the importance of multi-target compounds has been pointed out by several authors [6,13,91,100,101,102,103,104], since by having the ability to simultaneously modulate different targets, they may exhibit fewer side effects. Additionally, our group has previously verified that aromatase, ERα, and ERβ binding sites present some similarities, reinforcing the possible application of multi-target compounds in this disease [6].

However, for in vitro studies, we focused our attention on only 7 out of the 36 cannabinoids, CBDV, CBDB, CBDM, CBDA, CBCA, CBGVA, and CBC, as the remaining were not commercially available. Additionally, despite the absence of in silico evidence of a relevant modulation of aromatase and ERα, CBG and CBN were included in our study, as some studies in different tumors, including triple-negative breast cancer, have pointed out their therapeutic potential as anti-cancer compounds [75,76]. Regarding aromatase activity, we confirmed that CBG and CBN do not inhibit this enzyme. However, none of the other seven cannabinoids were able to greatly inhibit aromatase (only up to 12% inhibition). CBD, as expected and corroborating our previous work [65], presented high anti-aromatase activity. In fact, our previous molecular docking studies suggested the binding of CBD to key aromatase residues [65] and herein, CBD displayed the best docking score (−8.7 kcal/mol), followed by CBDV (−7.6 kcal/mol), which was found to inhibit aromatase by 12% at 10 μM. For aromatase inhibition, among the investigated cannabinoids, the presence of a carboxylic acid seems detrimental for activity (comparing, for example, CBG with CBGVA) and slight modifications in the hydrophobic tail of the cannabidiol type of metabolites influence the affinity for the enzyme, as evidenced when comparing CBDV (C₃) with its homologues CBDB (C₄) and CBDM (C₅).

In relation to ERα, different results were obtained for the nine studied phytocannabinoids. According to our transactivation results, cannabidiol derivatives CBDV, CBDB, and CBDM are ERα agonists, with CBDV and CBDB presenting full agonist activity properties (i.e., 75-fold increase over basal response [105]). The ER agonist activity verified for these cannabinoids is not in line with previous studies that, based on THC and CBD activities, indicated that cannabinoids do not exhibit ER agonist activity [62]. Although this agonist activity is not attractive for ER⁺ breast cancer cases, it may be clinically relevant in other pathological conditions, such as neurodegenerative disorders, liver injuries, or cardiovascular diseases, where a beneficial action should not be ruled out [106,107]. On the other hand, CBN, CBDA, CBCA, and CBC may potentially act as ER inverse agonists, which may be beneficial for ER⁺ breast cancer cases since, by presenting opposite actions to ER agonists, they prevent the tumorigenic effects of ERα. These results correlate with in silico studies in which CBDA (−8.3 kcal/mol) presented the highest affinity for the ER. For ER inverse agonism, cyclization, particularly chromene formation, was important for activity, as in the case of cannabichromene derivatives CBC and CBCA, and for chromane CBN. Moreover, except for the linear CBGVA, the carboxylic acid derivatives investigated (CBCA, CBDA) were active against this receptor.

Nevertheless, the behavior exhibited by the phytocannabinoids changes under hormone influence. Contrary to CBDV that loses ER agonistic activity, CBDB and CBDM become ER antagonists in the presence of E₂. In the presence of T, CBDA maintains ERα antagonist activity, while in the presence of E₂, this activity is abolished. These different behaviors can be explained by the binding affinities to ERα in the presence of the different hormones. Moreover, the fixed E₂ concentration (91.8 pM) used for the assessment of antagonistic activity induces the maximum activation of the receptor, which might be too high to detect activity of weaker antagonistic compounds. On the other hand, antagonistic activities in the presence of T (1 nM) might be more notable due to the concentration of T used, which is not associated with maximum effects. Curiously, CBDB fails as an ER antagonist in the presence of T. In both conditions, CBN and CBC act as ERα antagonists with inverse agonist properties, though this effect was only observed at the highest concentration for the latter compound. As previously reported, this behavior was similar to that of CBD [44]. This represents a therapeutic advantage for ER⁺ breast cancer and other clinical conditions that may be treated with ERα antagonists [108]. Of all the studied cannabinoids, only the two representatives of the cannabigerol type, the biosynthetic precursor CBGVA and CBG, showed no effect on ER activity, highlighting the importance of cyclization features for ER activity.

As previously mentioned, AR is gaining significant therapeutic attention in breast cancer, with some AR-target therapies being studied in different models. In fact, AR activation can modulate other receptors, such as ER and HER2 [109]. Regarding ER⁺ breast cancer and considering the beneficial effect of AR in those cases, the use of AR agonists would benefit the treatment of this disease, in a non-resistant scenario [20,87]. Unfortunately, our results, both in silico and in vitro, revealed that the nine cannabinoids studied acted as AR antagonists with inverse agonist properties, a behavior similar to the one observed for CBD [44]. This result is in accordance with some other studies that have pointed out that drugs of abuse, such as marijuana, by preventing the binding of dihydrotestosterone to AR, present AR antagonist activity [110,111]. Furthermore, due to the nature of this steroid receptor, almost only steroid hormones can act as AR agonists. Nevertheless, and despite the apparently disadvantageous effects of these compounds on AR, when ER is not overexpressed or activated in tumors, AR displays pro-survival and tumorigenic roles, contributing to tumor development [24,112]. This scenario is frequently found in resistant stages, which makes the use of AR antagonists an attractive approach. Thus, our data suggest that cannabinoids, by acting as AR antagonists with inverse agonist properties, might be a beneficial therapy in resistant scenarios, or might even impair the AR oncogenic role known for ER⁺ breast cancer cells treated with Exe [24]. In fact, there are some ongoing clinical trials with AR antagonists and AIs or anti-estrogens for breast cancer treatment [113,114]. Furthermore, by acting as AR antagonists, the studied cannabinoids may also be attractive for other pathological conditions where AR antagonists could be clinically applied, such as prostate cancer and benign prostatic hyperplasia [115,116]. Moreover, some triple-negative breast tumors show dependence on AR signaling for growth and survival [117]. In fact, two AR antagonists, Bicalutamide and Enzalutamide, were already evaluated in clinical trials (NCT00468715, NCT01889238) [118,119,120]. Therefore, the use of the minor phytocannabinoids evaluated in this study may also be beneficial for the management of this breast cancer subtype.

By using ToxPi metrics, we confirmed the previous assumption that CBD is the best cannabinoid when the simultaneous action towards aromatase, ER, and AR is considered, which highlights its therapeutic potential for ER⁺ breast cancer. The best minor phytocannabinoid is CBDB, a CBD derivative, mainly due to its great ER antagonistic activity over E₂ and its AR inverse agonistic and antagonistic activities. Similarly, the second best minor phytocannabinoid is CBN, whose score is also a result of its good scores on ER and AR molecular docking, but also on ER and AR antagonistic activities. Thus, considering that the effects exhibited by CBD on aromatase and ER surpass the ones induced on AR, which was not verified for the other cannabinoids, we can conclude that the scores attributed to the minor cannabinoids are mainly determined by the modulation of AR. Moreover, as formerly mentioned, ER antagonism is a crucial therapeutic action to be considered for ER⁺ breast cancer treatment, reason why we postulate that CBDB and CBN might be the most promising minor phytocannabinoids for hormone-dependent breast cancer treatment, along with the major phytocannabinoid CBD. Carboxylic acid derivatives, such as CBGVA and CBCA, occupy the last positions, indicating that this structural function is not beneficial overall. In the future, the study of these compounds in different breast cancer and non-cancer models will be of paramount importance to fully address the therapeutic potential of cannabinoids in breast cancer and guide their eventual application in the clinic.

4. Materials and Methods

5. Conclusions

Through in silico and in vitro assays, this study presents a comprehensive analysis of the potential therapeutic effects of the cannabinoids, described so far, for the treatment of hormone-dependent breast tumors. These compounds showed ability to modulate key targets responsible for tumor development, such as aromatase, ER, and AR, and the integrated activities implemented on them may together represent an advantage for treatment at different stages of the disease. In fact, the nine minor cannabinoids studied showed, in vitro, potential to act either as ER antagonists with inverse agonist properties, or as ER agonists. Moreover, despite the lack of significant aromatase inhibition, all cannabinoids may act as AR antagonists with inverse agonist action. Regarding their chemical characters, key features for ER antagonism like the chromene ring or detrimental substituents for aromatase inhibition, such as the carboxylic acid, were identified and can guide the selection of further investigations on minor cannabinoids. Moreover, by elucidating their molecular targets and mechanisms of action, this study opens up a prospective application of these compounds in other cancers and clinical conditions. From the best of our knowledge, this is the first study exploring the molecular targets of minor cannabinoids and, together with previous studies, it reinforces the importance and therapeutic potential of cannabinoids in breast cancer, paving the way for novel and alternative therapeutic approaches and highlighting the medicinal potential of Cannabis.

Acknowledgments

Abbreviations

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ph17091245/s1, Figure S1. Chemical structures of the cannabinoids described in Table 1. Figure S2. Chemical structures of the cannabinoids described in Table 2. Figure S3. Chemical structures of the cannabinoids described in Table 3. Figure S4. Molecular dynamics (MD) of ERα complexed with CBC (A), CBCA (B), CBD (C), CBDA (D), CBDB (E), CBDM (F), CBDV (G), CBG (H), CBGVA (I), and CBN (J). Results at the beginning (t = 0 ps, green) and at the end (t = 10,200 ps, blue) of the simulation (left), RMSD plot (center) and Potential Energy plot (right) during the time of simulation. Figure S5. Effects of the nine minor cannabinoids, CBG (A), CBDV (B), CBN (C), CBDB (D), CBDA (E), CBDM (F), CBCA (G), CBGVA (H) and CBC (I), on VM7LucE2 cell viability. Cells were treated with the cannabinoids (1, 5 and 10 μM) in the absence (ERα agonism) or presence (ERα antagonism) of testosterone (T; 1 nM) or estradiol (E2; 91.8 pM) for 24h. Results are presented as mean ± SEM of at least three independent experiments performed in triplicate. Statistically sig-nificant differences between control (cells not exposed to cannabinoids), set as 1, and cells not treated with T or E2 are expressed as * (p < 0.05), ** (p < 0.01) and *** (p < 0.001), while the differences between control and cells treated with cannabinoids in the presence of E2 are expressed as $ (p <0.05), $$ (p < 0.01) and $$$ (p < 0.001), and the differences between control cells and cells treated with cannabinoids in the presence of T are expressed as # (p < 0.05), ## (p < 0.01) and ### (p < 0.001). Figure S6. Effects of the nine minor cannabinoids, CBG (A), CBDV (B), CBN (C), CBDB (D), CBDA (E), CBDM (F), CBCA (G), CBGVA (H) and CBC (I), on CHO-K1 cells viability. Cells were treated with the cannabinoids (1, 5 and 10 μM) in the absence (AR agonism) or presence (AR an-tagonism) of R1881 (0.1 nM) for 24h. Results are presented as mean ± SEM of at least three inde-pendent experiments performed in triplicate. Statistically significant differences between control (cells not exposed to cannabinoids), set as 1, and cells not treated with R1881 are expressed as * (p < 0.05), ** (p < 0.01) and *** (p < 0.001), while the differences between control and cells treated with cannabinoids in the presence of R1881 are expressed as $$$ (p < 0.001).

Funding Statement

This research was funded by national funds from FCT—Fundação para a Ciência e a Tecnologia, in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences—UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB.

Author Contributions

C.F.A.: Writing—original draft, Investigation, Formal analysis, Validation, Methodology, Funding acquisition, Conceptualization. A.P.: Writing—review and editing, Formal analysis. M.J.V.: Writing—review and editing, Formal analysis, Resources. G.C.-d.-S.: Writing—review and editing, Supervision, Funding acquisition, Resources, Conceptualization. A.M.V.: Writing—review and editing, Resources. M.E.S.: Writing—review and editing, Resources. N.T.: Writing—review and editing, Supervision, Validation, Funding acquisition, Resources, Conceptualization. C.A.: Writing—review and editing, Formal analysis, Validation, Funding acquisition, Resources, Conceptualization, Supervision, Project administration. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors have no conflicts of interest to declare.

Footnotes

References