The Critical Role of Glutamine and Fatty Acids in the Metabolic Reprogramming of Anoikis-Resistant Melanoma Cells

Introduction: Circulating tumor cells (CTCs) represent the sub-population of cells shed into the vasculature and able to survive in the bloodstream, adhere to target vascular endothelial cells, and re-growth into the distant organ. CTCs have been found in the blood of most solid tumor-bearing patients and are used as a diagnostic marker. Although a complex genotypic and phenotypic signature characterizes CTCs, the ability to survive in suspension constitutes the most critical property, known as resistance to anoikis, e.g., the ability to resist apoptosis resulting from a loss of substrate adhesion. Here, we selected melanoma cells resistant to anoikis, and we studied their metabolic reprogramming, with the aim of identifying new metabolic targets of CTCs.Methods: Subpopulations of melanoma cells expressing a high anoikis-resistant phenotype were selected by three consecutive rocking exposures in suspension and studied for their phenotypic and metabolic characteristics. Moreover, we tested the efficacy of different metabolic inhibitors targeting glycolysis (2DG), LDHA (LDHA-in-3), the mitochondrial electron transport chain complex I (rotenone), glutaminase (BPTES), fatty acid transporter (SSO), fatty acid synthase (denifanstat), CPT1 (etomoxir), to inhibit cell survival and colony formation ability after 24 h of rocking condition.Results:Anoikis-resistant cells displayed higher ability to grow in suspension on agarose-covered dishes respect to control cells, and higher cell viability and colony formation capability after a further step in rocking condition. They showed also an epithelial-to-mesenchymal transition associated with high invasiveness and a stemness-like phenotype. Anoikis-resistant melanoma cells in suspension showed a metabolic reprogramming from a characteristic glycolytic metabolism toward a more oxidative metabolism based on the use of glutamine and fatty acids, while re-adhesion on the dishes reversed the metabolism to glycolysis. The treatment with metabolic inhibitors highlighted the effectiveness of rotenone, BPTES, SSO, and etomoxir in reducing the viability and the colony formation ability of cells capable of surviving in suspension, confirming the dependence of their metabolism on oxidative phosphorylation, using glutamine and fatty acids as the most important fuels.Discussion: This finding opens up new therapeutic strategies based on metabolic inhibitors of glutaminase and fatty acid oxidation for the treatment of CTCs and melanoma metastases.