Cannabinoids have been used as anti-emetic agents in cancer. However, multiple studies suggest that cannabinoids present important anti-tumor actions as well. Estrogen receptor-positive (ER⁺) breast cancer is the most diagnosed breast cancer subtype, and despite the success of endocrine therapy, endocrine resistance development is a major challenge, demanding the discovery or implementation of alternative therapeutic approaches. In line with this, and following our previous work, the benefits of combining the aromatase inhibitors (AIs) used in the clinic, anastrozole (Ana), letrozole (Let), and exemestane (Exe), with cannabinol (CBN) were evaluated. Experiments were performed in MCF-7aro cells and spheroids to assess activity against specific molecular targets and underlying mechanisms of action.

Among the three AIs studied, only the combination of CBN with Exe induced a significant beneficial impact on viability and growth of ER⁺ breast cancer cells and spheroids. Our results demonstrated that this combination was more effective than Exe in preventing the expression of aromatase and in modulating ERα and androgen receptor (AR) activity. In fact, the results revealed that CBN can prevent de novo synthesis of aromatase, surpass Exe’s weak estrogen-like effect, and avoid the unfavorable overexpression of AR. By comparing these two therapeutic strategies, as well as the previously studied combination of Exe plus cannabidiol (CBD), differential transcriptome profiles were detected, which may help to better understand the mechanism of action of cannabinoids and disclose their full potential in breast cancer treatment. In conclusion, this study strengthens the hypothesis that cannabinoids are important anti-cancer agents with attractive co-adjuvant properties.