DataSheet1_The critical role of glutamine and fatty acids in the metabolic reprogramming of anoikis-resistant melanoma cells.PDF

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.

引言：循环肿瘤细胞（Circulating Tumor Cells, CTCs）指脱落进入脉管系统、可在血流中存活、黏附于靶血管内皮细胞并在远处器官重新定植生长的细胞亚群。目前已在多数实体瘤患者的血液中检出CTCs，其可作为诊断标志物。尽管CTCs具有复杂的基因型与表型特征，但在悬浮状态下存活的能力是其最关键的属性，即失巢凋亡（anoikis）抗性——也就是抵抗因失去基质黏附而引发的细胞凋亡的能力。本研究筛选了具有失巢凋亡抗性的黑色素瘤细胞，并对其代谢重编程过程展开研究，旨在明确CTCs的新型代谢靶点。
方法：通过连续三次悬浮摇晃处理，筛选出表达高失巢凋亡抗性表型的黑色素瘤细胞亚群，并对其表型与代谢特征进行分析。此外，本研究测试了多种代谢抑制剂的效果，包括靶向糖酵解（glycolysis）的2DG、靶向乳酸脱氢酶A（LDHA）的LDHA-in-3、线粒体电子传递链复合物I抑制剂鱼藤酮（rotenone）、谷氨酰胺酶抑制剂BPTES、脂肪酸转运蛋白抑制剂SSO、脂肪酸合酶抑制剂地那司他（denifanstat）以及肉碱棕榈酰转移酶1（CPT1）抑制剂依托莫司（etomoxir），以评估它们在摇晃培养条件下处理24小时后对细胞存活与集落形成能力的抑制效果。
结果：相较于对照细胞，失巢凋亡抗性细胞在琼脂糖包被培养皿的悬浮环境中具有更强的增殖能力，且在进一步的摇晃培养条件下展现出更高的细胞活力与集落形成能力。这类细胞还呈现出与高侵袭性相关的上皮间质转化以及干细胞样表型。悬浮状态下的失巢凋亡抗性黑色素瘤细胞，其代谢模式从典型的糖酵解代谢转向以谷氨酰胺与脂肪酸为能源的氧化代谢；而当细胞重新黏附于培养皿表面时，代谢模式又会逆转回糖酵解状态。代谢抑制剂实验结果显示，鱼藤酮、BPTES、SSO与依托莫司可有效降低悬浮存活细胞的活力与集落形成能力，证实这类细胞的代谢依赖于氧化磷酸化，且以谷氨酰胺与脂肪酸作为主要能源物质。
讨论：本研究发现为基于谷氨酰胺酶与脂肪酸氧化代谢抑制剂的治疗策略提供了新方向，有望用于CTCs相关疾病与黑色素瘤转移的治疗。