ROLE OF THE CB1 CANNABINOID RECEPTOR IN BRAFV600E MELANOMA CELLS AND STEM-LIKE CELLS

Introduction: Human cutaneous melanoma is a skin cancer with high metastatic potential, enhanced heterogeneity, and resistance to chemotherapy. It has been recognized that one of the most important factors responsible for treatment failure is the presence of a small subpopulations of cancer cells, known as melanoma initiating cells, that have stem cell properties, tumorigenic potential and the ability to self-renew. Cannabinoid signaling regulates cell proliferation, differentiation and survival, with different outcomes depending on the molecular targets and cellular context involved. The aim of the current study is to investigate whether the endocannabinoid system have a role in determining the neoplastic phenotype of human melanoma cells. Methods: Human malignant melanoma cells, melanoma stem-like cells and normal human epidermal melanocytes were used as in vitro model system. Gene and protein expression were assessed by real-time PCR and western blotting. Knock-down of cannabinoid receptor type 1 (CB1) was performed in BRAFV600E mutant A375 cells using two independent CB1 short hairpin RNAs (shRNAs) (LV-shCB1-1 and LV-shCB1-2) and confirmed by real-time PCR and western blot. Analyses of cell growth, colony formation, cell cycle assay, migration capability and self-renewal ability were assessed in control (LV-c) and CB1-silenced cells. Results: Quantitative real-time PCR analyses showed that melanoma cells harboring BRAF V600E mutation expressed CB1 receptors, while BRAF-wild type cells, their correspondent melanoma stem-like cells, and normal melanocytes did not express or expressed CB1 at low levels. CB2 receptor expression was not found in the tested cell lines. Silencing of CB1 receptor in A375 cells induced a significant reduction in the proliferation potential, clonogenicity, migration and promotion G2/M phase arrest. Moreover, preliminary experiments revealed a decrease in self-renewal capacity of A375 stem-like cells. Conclusions: Our findings suggest a possible role of the endocannabinoid system, via CB1 receptors, in determining aggressive phenotype in melanoma cells. Experiments are ongoing in trying to elucidate the molecular mechanisms involved, particularly the relationship between CB1 receptor function and the BRAFV600E-mutated pathway.