# Molecular Targets of Minor Cannabinoids in Breast Cancer: In Silico and In Vitro Studies
## 1 Introduction
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 ER\(\lx@sectionsign\)55. More recently, our group has clarified the anti-cancer role of **CBD** and **THC**, and of the endocannabinoid anandamide (**AEA**) in MCF-7 aro cells, unveiling their mechanism of action and their ability to modulate key targets, such as aromatase, ER\(\alpha\), and ER\(\lx@sectionsign\)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 uM, 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 **\(\boldsymbol{\Delta^{9}}\)-THC** type, \(\boldsymbol{\Delta^{8}}\)-**THC** type, **CBD** type, cannabinoid (**CBG**) type, cannabinoid (**CBND**) type, cannabielsoin (**CBE**) type, cannabicyclol (**CBL**) type, cannabichromene (**CBC**) type, cannabinoid (**CBN**) type, cannabitriol (**CBT**) type, and miscellaneous type [66; 67; 27; 68; 69]. All the cannabinoids included in the different classes share a similar C21 terpenophenonic 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 cannabichromene acid (**CBCA**) were already addressed in diverse types of cancers, including breast [28; 45; 72; 73; 74; 75; 76; 77; 78]. 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, ER\(\alpha\), 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. Figure 2: Biosynthetic pathway of phytocannabinoid and phytocannabinoid class representation. Structural and biosynthetic relationships of the nine minor cannabinoids studied in vitro: cannabigerol (**CBG**), cannabigerol (**CBDV**), cannabinoid (**CBN**), cannabidiol-C4 (**CBDB**), cannabidiolic acid (**CBDA**), cannabidiol monomethyl ether (**CBDM**), cannabichromenic acid (**CBCA**), cannabigerovarinic acid (**CBGVA**), and cannabichromene (**CBC**).