# Molecular Targets of Minor Cannabinoids in Breast Cancer: In Silico and In Vitro Studies
## 3 Discussion
Curiously, **CBDB** fails as an ER antagonist in the presence of T. In both conditions, **CBN** and **CBC** act as ER\(\alpha\) 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 ER\(\alpha\) 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 E\({}_{2}\) 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.