# Molecular Targets of Minor Cannabinoids in Breast Cancer: In Silico and In Vitro Studies
## 3 Discussion  
The use of endocrine therapies, such as Als 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 [94; 95; 96; 13; 97; 98; 99; 14; 15; 16; 17; 18; 19; 13], 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 ER\(\alpha\) 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 ER\(\alpha\) signaling by up-regulating ER\(\beta\)[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 Als. 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 Als used in the clinic do bind to this key residue, thereby presenting high anti-aromatase activity [83; 97; 89]. ER\(\alpha\), 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 ER\(\alpha\) 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 ER\(\alpha\) 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 ER\(\alpha\) antagonists than as Als. Furthermore, 36 compounds out of the 45 potential Als and of the 85 potential ER antagonists were simultaneously considered as both promising Als as well as ER\(\alpha\) 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 [100; 101; 102; 103; 104; 6; 13], since by having the ability to simultaneously modulate different targets, they may exhibit fewer side effects. Additionally, our group has previously verified that aromatase, ER\(\alpha\), and ER\(\beta\) 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.