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

Abstract

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.

Author summary

Oxidation of long-chain fatty acids inside of the mitochondrial matrix provides an essential source of energy for some cells. Since long-chain fatty acids cannot freely pass into the mitochondrial matrix, they rely on a protein called carnitine palmitoyltransferase I (CPT1) for transport. Prior research has found that many tumors exhibit increased expression of CPT1 and/or sensitivity to CPT1 inhibition by a drug called etomoxir. These findings have led to thinking that cancer cells rely on fatty acid oxidation for energy. Here we present data that indicate otherwise, showing that inactivation of fatty acid oxidation has no effect on the proliferation of at least some cancer cell lines. Instead, these cells alter their utilization of other nutrients (such as glutamine) to compensate for the loss of fatty acid oxidation. We describe 2 discoveries that provide new insight into the role of fatty acid oxidation in cancer and help rationalize previous results. First, etomoxir has the off-target effect of inhibiting complex I of the electron transport chain. Second, CPT1 has other cellular functions that are independent of fatty acid oxidation. We suggest that one such function may be importing long-chain fatty acids into the mitochondria for anabolic fates, rather than catabolic oxidation.