The aryl-ureido fatty acid CTU activates endoplasmic reticulum stress and PERK/NOXA-mediated apoptosis in tumor cells by a dual mitochondrial-targeting mechanism

Highlights

• The ureido-fatty acid CTU rapidly targeted the mitochondrion in tumor cells.

• CTU selectively inhibited electron transport complex III and increased ROS.

• From RNA-seq, CTU activated the PERK/ER-stress pathway in vitro and in vivo

• Activation of the BH3-only protein NOXA disrupted the outer mitochondrial membrane.

• Dual-mitochondrial targeting agents like CTU are potential new anti-cancer therapeutics.

Abstract

The cancer cell mitochondrion is functionally different from that in normal cells and could be targeted to develop novel experimental therapeutics. The aryl-ureido fatty acid CTU (16({[4-chloro-3-(trifluoromethyl)phenyl]-carbamoyl}amino)hexadecanoic acid) is the prototype of a new class of mitochondrion-targeted agents that kill cancer cells. Here we show that CTU rapidly depolarized the inner mitochondrial membrane, selectively inhibited complex III of the electron transport chain and increased reactive oxygen species (ROS) production. From RNA-seq analysis, endoplasmic reticulum (ER)-stress was a major activated pathway in CTU-treated cells and in MDA-MB-231 tumor xenografts from CTU-treated nu/nu mice. Mitochondrion-derived ROS activated the PERK-linked ER-stress pathway and induced the BH3-only protein NOXA leading to outer mitochondrial membrane (OMM) disruption. The lipid peroxyl scavenger α-tocopherol attenuated CTU-dependent ER-stress and apoptosis which confirmed the critical role of ROS. Oleic acid protected against CTU-mediated apoptosis by activating Mcl-1 expression, which increased NOXA sequestration and prevented OMM disruption. Taken together, CTU both uncouples mitochondrial electron transport and activates ROS production which promotes ER-stress-dependent OMM disruption and tumor cell death. Dual-mitochondrial targeting agents like CTU offer a novel approach for development of new anti-cancer therapeutics.

Introduction

The tumor cell mitochondrion is a promising target for the development of novel anti-cancer therapeutics because it has a central role in cellular metabolism and apoptosis and because these processes are dysregulated in cancer cells [1]. The mitochondrial electron transport chain (ETC) generates ATP via multiprotein complexes located in the inner mitochondrial membrane (IMM; 2). Thus, ETC complexes I and II mediate electron transfer from NADH and FADH₂, respectively, to ubiquinol and then complex III. Complex III transfers electrons to cytochrome c and then to complex IV, which reduces molecular oxygen to water [3]. Complexes I, III and IV also pump protons into the mitochondrial intermembrane space, which establishes a proton gradient across the IMM that drives ATP synthesis by complex V (ATP-synthase). However, electron utilization and ATP production may be uncoupled by protonophoric agents that disrupt the IMM proton gradient [2].

The endoplasmic reticulum (ER) regulates the folding of newly synthesized proteins and their trafficking to subcellular destinations. However, damaged or misfolded proteins accumulate in the ER and promote ER-stress that activates the unfolded protein response (UPR) [4]. The UPR is a coordinated response mediated by the ER sensors activating transcription factor 6 (ATF6), protein kinase RNA-like ER kinase (PERK) and inositol-requiring enzyme 1 (IRE1). ATF6 increases the expression of chaperones to facilitate the egress of misfolded proteins from the ER, PERK activates eukaryotic translation initiation factor 2α (eIF2α) that inhibits further protein translation and IRE1 promotes RNA degradation and decreases protein synthesis [5]. Although these ER sensors coordinate mechanisms that attempt to relieve ER-stress, apoptosis is activated if the stress is extensive or prolonged [4].

It has been suggested that drug-mediated disruption of protein trafficking mechanisms could be utilized for the apoptotic elimination of cancer cells by novel agents [[6], [7], [8]]. The mitochondrion is a major source of reactive oxygen species (ROS) that regulate cell signaling and cell fate [9,10]. Such mitochondrion-derived stressors can activate ER-stress and the UPR [11]. PERK, but not the other ER-stress sensors, is expressed in mitochondria-associated ER membranes and functions as an important relay mechanism for the transmission of mitochondrial ROS death signals [12]. We reported previously that the novel aryl-ureido fatty acid CTU targeted the mitochondrion in MDA-MB-231 cells and uncoupled the ETC [13]. Most uncoupling agents, such as the triphenylphosphonium agents, are lipophilic cations that are attracted by the high membrane potential of the IMM and rapidly accumulate within the mitochondrial matrix [14]. In contrast, CTU is a protonophoric uncoupler that is negatively charged at physiological pH. After entry to the mitochondrion CTU relocates to the intermembrane space and transfers protons across the IMM into the matrix [13]. In addition to protonophoric uncoupling, CTU also activated apoptosis in cells in vitro and in xenografted nu/nu mice in vivo, although the mechanism is presently unknown [15]. To develop aryl-ureido fatty acids as potential anti-cancer agents detailed information on the underlying apoptotic mechanism is essential.

The present study was undertaken to define the mechanism by which the novel mitochondrion-targeted agent CTU activates apoptosis in cancer cells. The principal finding was that CTU inhibited mitochondrial ETC complex III and increased the formation of ROS that activated the PERK-mediated ER-stress pathway. This increased the expression of the BH3-only proapoptotic factor NOXA that promoted permeabilization of the OMM, which facilitated cytochrome c release and the activation of apoptosis. Together, the aryl-ureido fatty acid CTU has emerged as the prototype of a new class of potential therapeutics that promote tumor cell apoptosis by a novel dual-mitochondrial targeting mechanism involving complex III inhibition, increased ROS production and the activation of ER-stress in addition to ETC uncoupling.