Targeting ferroptosis in rhabdomyosarcoma cells.

Recent data suggest that rhabdomyosarcoma (RMS) cells might be vulnerable to oxidative stress-induced cell death. Here, we show that RMS are susceptible to cell death induced by Erastin, an inhibitor of the glutamate/cystine antiporter xc- that can increase reactive oxygen species (ROS) production via glutathione (GSH) depletion. Prior to cell death, Erastin caused GSH depletion, ROS production and lipid peroxidation. Importantly, pharmacological inhibitors of lipid peroxidation (i.e., Ferrostatin-1, Liproxstatin-1), ROS scavengers (i.e., α-Tocopherol, GSH) and the iron chelator Deferoxamine inhibited ROS accumulation, lipid peroxidation and cell death, consistent with ferroptosis. Interestingly, the broad-spectrum protein kinase C (PKC) inhibitor Bisindolylmaleimide I as well as the PKCα- and ß-selective inhibitor Gö6976 significantly reduced Erastin-induced cell death. Similarly, genetic knockdown of PKCα significantly protected RMS cells from Erastin-induced cell death. Furthermore, the broad-spectrum nicotinamide adenine dinucleotide phosphate-oxidase (NOX) inhibitor Diphenyleneiodonium and the selective NOX1/4 isoform inhibitor GKT137831 significantly decreased Erastin-stimulated ROS, lipid ROS and cell death. These data provide new insights into the molecular mechanisms of ferroptosis in RMS, contributing to the development of new redox-based treatment strategies.

Identification of Smac mimetics as novel substrates for p-glycoprotein.

Multidrug resistance (MDR) in cancer patients undergoing chemotherapy is preventing effective treatment of multiple cancer types including pediatric tumors. Resistance to chemotherapeutic drugs in cancer cells is frequently associated with high expression of p-glycoprotein, a transporter in the plasma membrane that can mediate cellular drug export. Here, we generated pediatric cancer cells with acquired resistance to the chemotherapeutic drug vincristine (VCR). In these cells, acquired resistance is associated with increased expression of p-glycoprotein. VCR-resistant cells display an MDR phenotype and have acquired resistance to multiple other chemotherapeutic drugs including doxorubicin (DOXO) and etoposide (ETO). Notably, we discovered that these cells also display cross-resistance with several Smac mimetics, a novel class of experimental cancer therapeutics designed to induce apoptosis by inhibiting Inhibitor of Apoptosis (IAP) proteins. Resistance to Smac mimetics is reversible in the presence of p-glycoprotein inhibitors, highlighting Smac mimetics as novel substrates for p-glycoprotein. The identification of Smac mimetics as substrates for p-glycoproteins may influence the design of future clinical trials to prevent usage of Smac mimetics in the context of MDR or, alternatively, combine Smac mimetics with p-glycoprotein inhibitors to maximize their efficiency.

Targeting redox homeostasis in rhabdomyosarcoma cells: GSH-depleting agents enhance auranofin-induced cell death.

Rhabdomyosarcoma (RMS) cells have recently been reported to be sensitive to oxidative stress. Therefore, we investigated whether concomitant inhibition of the two main antioxidant defense pathways, that is, the thioredoxin (TRX) and the glutathione (GSH) systems, presents a new strategy to trigger cell death in RMS. In this study, we discover that GSH-depleting agents, i.e. γ-glutamylcysteine synthetase inhibitor, buthionine sulfoximine (BSO) or the cystine/glutamate antiporter inhibitor erastin (ERA), synergize with thioredoxin reductase (TrxR) inhibitor auranofin (AUR) to induce cell death in RMS cells. Interestingly, AUR causes accumulation of ubiquitinated proteins when combined with BSO or ERA, in line with recent reports showing that AUR inhibits the proteasome besides TrxR. Consistently, AUR/BSO or AUR/ERA cotreatment increases ubiquitination and expression of the short-lived proteins NOXA and MCL-1, accompanied by increased binding of NOXA to MCL-1. Notably, NOXA knockdown significantly rescues RMS cells from AUR/BSO- or AUR/ERA-induced cell death. In addition, AUR acts together with BSO or ERA to stimulate BAX/BAK and caspase activation. Of note, BSO or ERA abolish the AUR-stimulated increase in GSH levels, leading to reduced GSH levels upon cotreatment. Although AUR/BSO or AUR/ERA cotreatment enhances reactive oxygen species (ROS) production, only thiol-containing antioxidants (i.e., N-acetylcysteine (NAC), GSH), but not the non-thiol-containing ROS scavenger α-Tocopherol consistently suppress AUR/BSO- and AUR/ERA-stimulated cell death in both cell lines. Importantly, re-supply of GSH or its precursor NAC completely prevents AUR/ERA- and AUR/BSO-induced accumulation of ubiquitinated proteins, NOXA upregulation and cell death, indicating that GSH depletion rather than ROS production is critical for AUR/BSO- or AUR/ERA-mediated cell death. Thus, by demonstrating that GSH-depleting agents enhance the antitumor activity of AUR, we highlight new treatment options for RMS by targeting the redox homeostasis.