Oncogene (2015) 34, 3296–3304 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc ORIGINAL ARTICLE Isoprenylcysteine carboxylmethyltransferase regulates mitochondrial respiration and cancer cell metabolism JT Teh1,5, WL Zhu1,2,5, OR Ilkayeva3,YLi4, J Gooding3, PJ Casey1, SA Summers4, CB Newgard3 and M Wang1,2 Isoprenylcysteine carboxylmethyltransferase (Icmt) catalyzes the last of the three-step posttranslational protein prenylation process for the so-called CaaX proteins, which includes many signaling proteins, such as most small GTPases. Despite extensive studies on Icmt and its regulation of cell functions, the mechanisms of much of the impact of Icmt on cellular functions remain unclear. Our recent studies demonstrated that suppression of Icmt results in induction of autophagy, inhibition of cell growth and inhibition of proliferation in various cancer cell types, prompting this investigation of potential metabolic regulation by Icmt. We report here the findings that Icmt inhibition reduces the function of mitochondrial oxidative phosphorylation in multiple cancer cell lines. In-depth oximetry analysis demonstrated that functions of mitochondrial complex I, II and III are subject to Icmt regulation. Consistently, Icmt inhibition decreased cellular ATP and depleted critical tricarboxylic acid cycle metabolites, leading to suppression of cell anabolism and growth, and marked autophagy. Several different approaches demonstrated that the impact of Icmt inhibition on cell proliferation and viability was largely mediated by its effect on mitochondrial respiration. This previously unappreciated function of Icmt, which can be therapeutically exploited, likely has a significant role in the impact of Icmt on tumorigenic processes. Oncogene (2015) 34, 3296–3304; doi:10.1038/onc.2014.260; published online 25 August 2014 INTRODUCTION These phenotypes suggested to us that the cells in which Icmt is Prenylation is a three-step posttranslational lipid modification inhibited might be metabolically compromised, as impairment of process in the maturation of a number of proteins involved in cell ATP production induces a starvation response leading to similar regulation. The majority of prenylated proteins belong to a group presentations. We therefore investigated the impact of Icmt termed ‘CaaX proteins’ that are defined by a specific C-terminal inhibition on cell respiration, energy status and metabolism. Our amino-acid sequence ‘cysteine-aliphatic-aliphatic-any’, serving as findings demonstrate that Icmt has a previously unappreciated fi the consensus sequence for their modification. Following role in cellular metabolism, which account signi cantly for its isoprenoid addition on the cysteine and proteolytic removal of impact on growth and proliferation and its role in tumorigenesis. the –aaX sequence, the final step is methylation of the C-terminal prenylcysteine by isoprenylcysteine carboxylmethyltransferase (Icmt).1,2 The prenylation process is required for proper function RESULTS of the modified protein, either as a mediator of specific subcellular Icmt inhibition leads to AMPK activation, a result of energy localization, a determinant for specific protein–protein interac- depletion 3–5 tions, or protein stability. Following the discovery that K-Ras We have shown previously that treatment of multiple cancer cell is significantly mislocalized in cells that lack the Icmt types, including PC3 prostate and MDA-MB-231 breast cancer 4 methyltransferase, and that the abilities of K-Ras to transform cells, with a small-molecule Icmt inhibitor termed cysmethynil led fibroblasts and promote myeloproliferative disease and lung to inhibition of the mammalian target of rapamycin (mTOR) and cancer are dependent on Icmt,6,7 this enzyme has gained elevated autophagy.8,9 This Icmt inhibitor-induced autophagy attention as a potential cancer target. However, it is increasingly phenotype was extensively investigated in our prior studies, which recognized that the impact of Icmt-catalyzed CAAX protein demonstrated consistent increased LC3 I to LC3 II conversion and methylation is not limited to processes mediated by Ras; this increased autophagy flux measured by multiple modalities, such carboxylmethylation also affects basic cell functions mediated by as baflomycin cotreatment and colocalization of RFP-LC3- and CaaX proteins other than Ras.2 Unravelling the roles of Icmt in GFP-LC3-positive vesicles. However, the mechanism through regulating CaaX protein-driven processes is fundamentally which Icmt inhibition impacts this important biologic response important. was unresolved. To investigate the impact of cysmethynil Pharmacologic or genetic suppression of Icmt results in slow treatment upstream of mTOR, we assessed the activation status cell growth, cell cycle arrest and excessive autophagy, of possible regulators including AMP-dependent kinase (AMPK). which account for the anticancer efficacies of Icmt inhibition.8,9 AMPK activity responds to ATP levels and hence provides a direct 1Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore, Singapore; 2Department of Biochemistry, National University of Singapore, Singapore, Singapore; 3Sarah W Stedman Nutrition and Metabolism Center, and Duke Institute of Molecular Physiology, Duke University Medical Center, Durham, NC, USA and 4Program in Cardiovascular and Metabolic Disorders, Duke-NUS Graduate Medical School, Singapore, Singapore. Correspondence: Dr M Wang, Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, 8 College Road, Singapore 169857, Singapore. E-mail: [email protected] 5These authors contributed equally to this work. Received 4 February 2014; revised 9 June 2014; accepted 3 July 2014; published online 25 August 2014 Icmt regulates mitochondrial respiration JT Teh et al 3297 gauge of cellular energy status. Once activated, AMPK suppresses cysmethynil-treated PC3 cells (Figure 1d). Although ATP is anabolic activities, activates fuel catabolism and promotes considered the major energy currency in cells, it is important to autophagy to increase energy stores.10,11 measure the other nucleoside triphosphates in the evaluation of Indeed, the treatment of PC3 cells with cysmethynil elevated cell energy status, as there exists a dynamic balance between levels of phosphorylated AMPK in a dose-dependent manner different NTPs, energy currency molecules.12,13 The reduction of (Figure 1a). We also observed increased inhibitory phosphoryla- NTPs in cysmethynil-treated cells suggests that AMPK activation is tion of acetyl-coA carboxylase (ACC) at serine 79, an AMPK likely the result of energy deficiency. It is worth noting that the phosphorylation site, consistent with increased AMPK activity. energy deplete state and associated signaling changes induced by Aligned with our previous reports of autophagy induction, there Icmt inhibition is not limited to PC3 cells; MDA-MB-231 cells was also a dose-dependent accumulation of the autophagy responded in a similar manner (Supplementary Figure 1), marker LC3 II, which paralleled the response of AMPK activation suggesting a general regulatory mechanism by Icmt in cell energy (Figure 1a). Similar phenotype of elevated pAMPK level was metabolism. observed upon small interfering RNA (siRNA) suppression of Icmt expression (Figure 1b). Further, we subjected wild-type mouse Cysmethynil treatment reduces mitochondrial respiratory capacity embryonic fibroblast (MEF) cells and Icmt-null MEF cells to To investigate the cause of cysmethynil-induced energy depletion, cysmethynil treatment. While Icmt-null MEFs exhibit higher basal we studied mitochondrial function in the cells. Cysmethynil- pAMPK and LC3 II levels, robust increases in pAMPK and LC3 II treated cells exhibit markedly reduced basal/resting-respiration levels are only observed in the wild-type MEFs upon cysmethynil rates (Figure 2a). In addition, cysmethynil-treated cells exhibited a treatment (Figure 1c). These genetic suppression studies provided shallower drop in oxygen consumption rate (OCR) upon the compelling evidence that effect of cysmethynil on AMPK addition of oligomycin, an ATPase inhibitor, in comparison with activation and autophagy is Icmt dependent. the untreated cells (Figure 2a), suggesting attenuated ATP The parallel elevation of autophagy and activation of AMPK are production before oligomycin addition. This result is consistent indications that Icmt-induced autophagy is mediated by AMPK with the NTP quantification study and the phenotype of AMPK activation, which can be either a direct response to cellular energy activation shown above. Trifluorocarbonylcyanide phenylhydra- status or a result of modulation by upstream molecules. In the zone (FCCP), uncoupling the electron transport system from investigation for possible etiology of Icmt-inhibition-mediated oxidative phosphorylation, is often used to assess the maximal AMPK activation, we analyzed the levels of nucleotide tripho- respiratory capacity of cells. Cysmethynil-treated cells displayed sphates (NTPs) in cysmethynil-treated and control cells. Significant lower FCCP-induced respiration in comparison with untreated decreases in levels of ATP, GTP, CTP and UTP were observed in cells (Figure 2a), indicative of reduction in the maximal respiratory capacity and potential rate for ATP production. Last, the differences in respiration between cysmethynil-treated and control cells are not likely from non-mitochondrial oxygen PC3 1.2 fi Cysm consumption, as no signi cant differences