Identifying off-target effects of etomoxir reveals that carnitine palmitoyltransferase I is essential for cancer cell proliferation independent of β-oxidation

It has been suggested that some cancer cells rely upon fatty acid oxidation (FAO) for energy. Here we show that when FAO was reduced approximately 90% by pharmacological inhibition of carnitine palmitoyltransferase I (CPT1) with low concentrations of etomoxir, the proliferation rate of various cancer cells was unaffected. Efforts to pharmacologically inhibit FAO more than 90% revealed that high concentrations of etomoxir (200 μM) have an off-target effect of inhibiting complex I of the electron transport chain. Surprisingly, however, when FAO was reduced further by genetic knockdown of CPT1, the proliferation rate of these same cells decreased nearly 2-fold and could not be restored by acetate or octanoic acid supplementation. Moreover, CPT1 knockdowns had altered mitochondrial morphology and impaired mitochondrial coupling, whereas cells in which CPT1 had been approximately 90% inhibited by etomoxir did not. Lipidomic profiling of mitochondria isolated from CPT1 knockdowns showed depleted concentrations of complex structural and signaling lipids. Additionally, expression of a catalytically dead CPT1 in CPT1 knockdowns did not restore mitochondrial coupling. Taken together, these results suggest that transport of at least some long-chain fatty acids into the mitochondria by CPT1 may be required for anabolic processes that support healthy mitochondrial function and cancer cell proliferation independent of FAO.已有研究表明，部分癌细胞依赖脂肪酸氧化（fatty acid oxidation, FAO）获取能量。本研究显示，当通过低浓度依托莫司（etomoxir）对肉碱棕榈酰转移酶I（carnitine palmitoyltransferase I, CPT1）进行药理学抑制，使脂肪酸氧化水平降低约90%时，多种癌细胞的增殖速率未受影响。尝试通过药理学手段将脂肪酸氧化抑制程度提升至90%以上时发现，高浓度依托莫司（200 μM）存在脱靶效应，可抑制电子传递链（electron transport chain）的复合物I（complex I）。令人意外的是，若通过基因敲低CPT1进一步降低脂肪酸氧化水平，上述癌细胞的增殖速率会下降近一倍，且无法通过补充乙酸盐或辛酸恢复。此外，CPT1敲低细胞的线粒体形态（mitochondrial morphology）发生改变，线粒体偶联（mitochondrial coupling）功能受损；而经依托莫司抑制约90%CPT1活性的细胞则无此变化。对CPT1敲低细胞分离的线粒体进行脂质组学分析（lipidomic profiling）显示，其线粒体中结构脂质与信号脂质的浓度均有所降低。此外，在CPT1敲低细胞中表达催化失活的CPT1，无法恢复其线粒体偶联功能。综上，上述结果表明，CPT1介导的至少部分长链脂肪酸向线粒体的转运，可能参与支持健康线粒体功能与癌细胞增殖的合成代谢过程（anabolic processes），且该过程不依赖于脂肪酸氧化。