Author response: Pharmacological inhibition of cystine–glutamate exchange induces endoplasmic reticulum stress and ferroptosisFull text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Exchange of extracellular cystine for intracellular glutamate by the antiporter system xc− is implicated in numerous pathologies. Pharmacological agents that inhibit system xc− activity with high potency have long been sought, but have remained elusive. In this study, we report that the small molecule erastin is a potent, selective inhibitor of system xc−. RNA sequencing revealed that inhibition of cystine–glutamate exchange leads to activation of an ER stress response and upregulation of CHAC1, providing a pharmacodynamic marker for system xc− inhibition. We also found that the clinically approved anti-cancer drug sorafenib, but not other kinase inhibitors, inhibits system xc− function and can trigger ER stress and ferroptosis. In an analysis of hospital records and adverse event reports, we found that patients treated with sorafenib exhibited unique metabolic and phenotypic alterations compared to patients treated with other kinase-inhibiting drugs. Finally, using a genetic approach, we identified new genes dramatically upregulated in cells resistant to ferroptosis. https://doi.org/10.7554/eLife.02523.001 eLife digest Sugars, fats, amino acids, and other nutrients cannot simply diffuse into the cell. Rather, they must be transported across the cell membrane by specific proteins that stretch from one side of the cell membrane to the other. One such ‘transporter’—system xc−—is of special interest. This transporter imports one molecule of cystine from outside the cell in exchange for one molecule of glutamate from inside the cell. Cystine, a variant of the amino acid cysteine, is essential for synthesizing new proteins and for preventing the accumulation of toxic species inside the cell. Not surprisingly, many cancer cells are dependent upon the transport activity of system xc− for growth and survival. Drugs that can inhibit system xc− could therefore be part of potential treatments for cancer and other diseases. Dixon, Patel, et al. have found that the compound erastin is a very effective inhibitor of system xc− function. Certain versions of erastin are over 1000 times more potent than the previously known best inhibitor of system xc−, sulfasalazine. Dixon, Patel et al. found that using erastin and sulfasalazine to inhibit system xc− in cancer cells grown in a petri dish results in an unusual type of iron-dependent cell death called ferroptosis. By inhibiting the uptake of cystine, erastin and other system xc− inhibitors interfere with the cellular machinery that folds proteins into their final, three-dimensional shape. The accumulation of these partially-folded proteins in the cell causes a specific kind of cellular stress that can be used as a readout, or biomarker, for the inhibition of system xc−. Such a biomarker will be essential for identifying cells in the body that have been exposed to agents that inhibit system xc− and that are undergoing ferroptosis. Unexpectedly, Dixon, Patel et al. also found that the FDA-approved anti-cancer drug sorafenib inhibits system xc−. Other drugs in the same class as sorafenib do not share this unusual property. Dixon, Patel, et al. synthesized variants of sorafenib and identified sites on the drug that are necessary for it to be able to interfere with system xc−. Alongside the erastin derivatives, these new molecules may help to develop new drugs that can inhibit this important transporter in a clinical setting. https://doi.org/10.7554/eLife.02523.002 Introduction Transporters for small molecule nutrients, including sugars, nucleotides, and amino acids, are essential for cellular metabolism and represent potential targets for drug development (Hediger et al., 2013). System xc− is a cell-surface Na+-independent cystine–glutamate antiporter composed of the 12-pass transmembrane transporter protein SLC7A11 (xCT) linked via a disulfide bridge to the single-pass transmembrane regulatory subunit SLC3A2 (4F2hc) (Sato et al., 1999; Conrad and Sato, 2012). System xc− is required for normal mammalian blood plasma redox homeostasis, skin pigmentation, immune system function, and memory formation (Chintala et al., 2005; Sato et al., 2005; De Bundel et al., 2011). Aberrant system xc− function is implicated in tumor growth and survival, cancer stem cell maintenance, drug resistance, and neurological dysfunction (Okuno et al., 2003; Buckingham et al., 2011; Ishimoto et al., 2011; Yae et al., 2012; Timmerman et al., 2013); inhibition of system xc− may prove useful in a number of therapeutic contexts. Efforts to treat gliomas and lymphomas in human patients by modulating system xc− activity with the low potency, metabolically unstable small molecule, sulfasalazine (SAS, Gout et al., 2001), were unsuccessful (Robe et al., 2009). While some progress has been made toward developing more potent compounds based on the SAS scaffold (Shukla et al., 2011), the identification of system xc− inhibitors based on alternative scaffolds remains a pressing need and would be useful to test the hypothesis that system xc− inhibition is therapeutically beneficial in glioma and other contexts. We previously demonstrated that the small molecule erastin prevents Na+-independent cystine uptake (Dixon et al., 2012), suggesting that erastin may inhibit system xc− function and represent a novel scaffold targeting this transport system. Intriguingly, treatment of some cell lines with erastin or SAS triggers an iron-dependent, non-apoptotic form of cell death, termed ferroptosis (Dixon et al., 2012). Ferroptosis is characterized by the accumulation of intracellular soluble and lipid reactive oxygen species (ROS), a process that is counteracted by the glutathione-dependent activity of the enzyme glutathione peroxidase 4 (GPX4) (Dixon and Stockwell, 2013; Yang et al., 2014). Erastin, and other ferroptosis-inducing compounds of this class, are therefore of interest both for their effects on amino acid transport and their ability to induce a novel cell death pathway. In this study, we show that erastin and its analogs specifically inhibit cystine uptake via system xc−, trigger ferroptosis in a variety of cellular contexts and act much more potently than SAS. Surprisingly, we found that the clinically approved multi-kinase inhibitor sorafenib can also inhibit system xc− and trigger ferroptosis under some conditions, an observation that may be relevant to both the anti-cancer properties and the profile of adverse events associated with this drug. We further show that small molecule inhibition of system xc− function leads to endoplasmic reticulum (ER) stress, as indicated by the transcriptional upregulation of genes linked to the ER stress response. The upregulation of the ER stress response gene CHAC1 (ChaC, cation transport regulator homolog 1) serves as a useful pharmacodynamic marker of system xc− inhibition. Finally, we found that resistance to system xc− inhibition is correlated with dramatically increased expression of AKR1C family members that regulate the detoxification of oxidative lipid breakdown products, providing potential insight into the downstream consequences of system xc− inhibition, and the execution mechanism of ferroptosis. Results Consistent induction of ferroptosis in various cells under a variety of growth conditions Erastin and SAS were previously shown to trigger ferroptosis in human HT-1080 fibrosarcoma cells grown on two-dimensional substrates with atmospheric levels of oxygen (i.e., 21% oxygen) (Dixon et al., 2012). We endeavored to generalize and validate the lethality of erastin towards cancer cells in several ways. First, we tested whether the same effects were observed in other cell types using a ‘modulatory profiling’ strategy (Wolpaw et al., 2011; Dixon et al., 2012). This method allows for the simplified detection and presentation of small molecule combination effects on cell viability (modulatory effect, Me < 0, sensitization; Me = 0, no effect; Me > 0, rescue). We observed that in five different human cancer cell lines, cell death induced by either erastin or SAS was rescued by the same canonical ferroptosis inhibitors: the iron chelator ciclopirox olamine (CPX), the lipophilic antioxidants trolox and ferrostatin-1 (Fer-1), the MEK inhibitor U0126, the protein synthesis inhibitor cycloheximide (CHX) and the reducing agent beta-mercaptoethanol (β-ME) (Dixon et al., 2012; Figure 1A,B). Thus, the ferroptotic death phenotype, whether induced by erastin or SAS, was similar in all cell lines tested. The inhibition of cell death by β-ME indicates that cell death most likely involves inhibition of system xc− function, as β-ME treatment can generate mixed disulfides taken up by other transporters, thereby circumventing the need for system xc− function (Ishii et al., 1981). Figure 1 Download asset Open asset Cell death is triggered by erastin and related compounds in different cell lines under a variety of physiological conditions. (A and B) Modulatory effect (Me) profiles of erastin- and SAS-induced death in five different cell lines (143B, BJeHLT, BJeLR, Calu-1, and HT-1080) in response to six different cell death inhibitors (U0126, Trolox, Fer-1, CPX, CHX, β–ME) or the vehicle DMSO. Me >0 indicates rescue from cell death. (C and D) Relative viability of MCTSs formed over 72 hr from HT-1080 (C) or Calu-1 (D) cells in response to erastin, RSL3 or staurosporine (STS) ±β-ME or ferrostatin-1 (Fer-1). Viability was assessed by Alamar blue and represents mean ± SD from three independent biological replicate experiments. Data were analyzed by two-way ANOVA with Bonferroni post-tests, *p<0.05, **p<0.05, ***p<0.001, ns = not significant. (E and F) Viability of HT-1080 (E) and DU145 (F) cells cultured under 1% or 21% O2 levels in response to erastin (5 μM) ±Fer-1 (1 μM) or CPX (5 μM). Viability was assessed by Alamar blue and represents mean ± SD from three independent biological replicate experiments. https://doi.org/10.7554/eLife.02523.003 Next, we sought to test whether the lethal mechanisms of action of erastin and SAS were influenced by cell growth architecture. Specifically, we tested whether the ferroptotic lethal mechanism could be activated in multicellular tumor spheroids (MCTSs), three-dimensional cellular aggregates proposed to recapitulate key aspects of the structural and metabolic heterogeneity observed in tumor fragments and micrometastases (Friedrich et al., 2009). We grew MCTSs from HT-1080 and Calu-1 cells for 72 hr and then investigated the effects of erastin ±β-ME or ±Fer-1 on MCTS growth and viability. For comparison, we also tested the growth inhibitory effects of (1S, 3R)-RSL3 (hereafter RSL3), a small molecule that triggers ferroptosis by inhibiting GPX4, which is downstream of system xc− in the ferroptotic cascade (Yang et al., 2014), as well as staurosporine (STS), which triggers apoptosis. We observed that HT-1080 and Calu-1 MCTSs were killed by erastin and RSL3 (Figure 1C,D). The effects of both erastin and RSL3 were rescued by Fer-1, while β-ME suppressed the lethality of erastin, but not of RSL3, as expected (Figure 1C,D). Neither β-ME nor Fer-1 modulated the effects of STS on MCTS growth or viability (Figure 1C,D). These observations indicate that erastin, as well as RSL3, are able to trigger ferroptosis in a similar manner in both two- and three-dimensional culture conditions. Finally, given that erastin triggers an oxidative form of cell death, we tested whether the lethality of erastin was affected by growth in low (1%) vs high (21%) levels of O2. Cells from two different erastin-sensitive cancer cell lines (HT-1080 and DU-145) were grown for 24 hr under low or high O2 levels and then treated for a further 24 hr with various agents, prior to the analysis of cell death. We observed that compared to DMSO-treated cells, erastin (5 μM)-treated cells were killed under both high and low O2 conditions with little (DU-145) or no (HT-1080) difference in lethality (Figure 1E,F). In both cell lines, erastin-induced death was suppressed by both Fer-1 (1 μM) and CPX (5 μM) (Figure 1E,F), indicating that the same lethal mechanism (i.e., ferroptosis) was responsible for cell death under both high and low O2 conditions. Thus, even under relatively low O2 conditions, it is still possible for erastin to activate the ferroptotic mechanism. Erastin inhibits system xc− function potently and specifically The ability to modulate system xc− activity may be clinically useful, but requires small molecule inhibitors with suitable pharmacological properties that are also specific for this antiporter (Gorrini et al., 2013). Erastin treatment (5 μM) completely abolished the Na+-independent uptake of radiolabelled [14C]-cystine in both HT-1080 fibrosarcoma and Calu-1 lung carcinoma cancer cells, as did sulfasalazine (SAS) at 100-fold higher concentrations (500 μM) (Figure 2A). Conversely, erastin and SAS had no effect on Na+-independent [14C]-phenylalanine uptake (Figure 2B). An excess of cold D-phenylalanine did suppress [14C]-phenylalanine uptake, confirming that Phe transport was inhibitable under these assay conditions (Figure 2B). Thus, in HT-1080 and Calu-1 cells, erastin and SAS block system xc− (SLC7A11 + SLC3A2)-mediated cystine uptake selectivity over other transport systems and amino acids, such as system-L-(SLC7A5 + SLC3A2)-mediated Phe uptake. Figure 2 with 1 supplement see all Download asset Open asset Erastin inhibits system xc− function potently and specifically. (A and B) Na+-independent uptake of 14C-cystine (A) and 14C-L-phenylalanine (Phe) (B) over 5 min in HT-1080 and Calu-1 cells treated with erastin or SAS. D-Phe was included as a positive control in B. (C) Structure and lethal potency (EC50 in HT-1080 cells) of erastin and the inactive erastin analog erastin-A8. (D) Dose-dependent inhibition of glutamate release by erastin and erastin-A8 (Era-A8). (E) Glutamate release ±erastin in HT-1080 cells in which SLC7A11 was silenced for 48 hr using two independent siRNAs. (F) SLC7A11 mRNA levels assayed using RT-qPCR in si-SLC7A11-transfected cells. (G) Glutamate release in response to erastin, SAS, RSL3 artesunate and PEITC, ±beta-mercaptoethanol (β-ME). (H) Dose-response analysis of glutamate release from HT-1080 and Calu-1 cells in response to erastin and SAS. All data are from three independent biological replicates. Data are presented as mean ± SD. Data in A and B are normalized to DMSO controls (set to 100%). Data in A, B, E and G were analyzed by ANOVA with Bonferroni post-tests, *p<0.05, ***p<0.001, ns = not significant. https://doi.org/10.7554/eLife.02523.004 We confirmed the ability of erastin and SAS to inhibit system xc− using an enzyme-coupled fluorescent assay that detects glutamate release into Na+-containing culture medium (Figure 2—figure supplement 1A). We validated this assay in three ways. First, we showed that erastin (1) inhibited glutamate release, while a non-lethal (Yagoda et al., 2007) erastin analog lacking the p-chlorophenoxy moiety (erastin-A8, 2) did not (Figure 2C,D). Second, we showed that both erastin treatment and silencing of SLC7A11 with either of two independent siRNAs resulted in a significant, quantitatively similar inhibition of glutamate release (Figure 2E,F); silencing of the system L transporter subunit SLC7A5 using two independent siRNAs had no effect on basal or erastin-mediated inhibition of glutamate release (Figure 2—figure supplement 1B,C). Third, we found that only erastin and SAS inhibited glutamate release, while, as expected, RSL3, artesunate and PEITC did not; while artesunate and PEITC induce iron-dependent cell death, neither are known to inhibit system xc− or induce ferroptosis (Trachootham et al., 2006; Hamacher-Brady et al., 2011; Dixon et al., 2012; Figure 2G). Thus, the above results suggest that both erastin and SAS specifically inhibit SLC7A11-dependent system xc− function. The ability of erastin to specifically inhibit cystine uptake via system xc− is further supported by recent metabolomic profiling data (Skouta et al., 2014; Yang et al., 2014) and gene expression experiments described below. In light of disappointing clinical results using SAS (Robe et al., 2009), it is desirable to identify potent inhibitors of system xc− with favorable pharmacological properties. Using the glutamate release assay to quantify inhibition of system xc− activity, we found that erastin was ∼2500 times more potent than SAS as an inhibitor of system xc− function in both HT-1080 and Calu-1 cells (HT-1080: erastin IC50 = 0.20 µM, 95% C.I. 0.11–0.34 µM, SAS IC50 = 450 µM, 95% C.I. 280–710 µM; Calu-1: erastin IC50 = 0.14 µM, 95% C.I. 0.081–0.21 µM, SAS IC50 = 460 µM, 95% C.I. 350–590 µM) (Figure 2H). Thus, the erastin scaffold may afford a more suitable starting point than SAS for the development of potent and selective inhibitors of system xc− function. Erastin structure-activity relationship (SAR) analysis and isolation of analogs with improved potency We hypothesized that it would be possible to improve further the potency of the erastin scaffold through targeted synthesis. Towards this end, we undertook a search for more potent analogs, beginning with an achiral analog (3, Yang et al., 2014) that lacked the methyl group at the chiral center, and that had an isoproproxy substituent in place of the ethoxy group on erastin (1) (Figure 3A). This compound (3) was more synthetically accessible, but otherwise exhibited a similar lethal potency as erastin in HT-1080 cells. We synthesized 19 analogs of 3 (Supplementary file 1, Figure 3A. Data also available as ‘Extended Materials and Methods’ from Dryad data Repository [Dixon et al., 2014]), and tested each in HT-1080 cells in a 10-point, twofold dose-response assay for lethal potency and efficacy (Figure 3B). To assess in each case whether lethality involved inhibition of system xc− and induction of ferroptosis, as opposed to the induction of another form of death, experiments were performed ±β-ME. To assess in a parallel assay the correlation between lethal potency and inhibition of system xc− activity, we examined glutamate release using a high throughput, 96-well Amplex Red assay system in human CCF-STTG1 astrocytoma cells (Figure 3B). Overall, the lethal potency in HT-1080 cells was found to correlate significantly with the degree of system xc− inhibition observed in CCF-STTG1 cells (Pearson R2 = 0.86, p<0.0001). Of note, we observed that glutamate release in CCF-STTG1 cells was in general more sensitive to erastin and analogs than HT-1080 cells (compare Figure 3B to Figure 2H). These results support the hypothesis that the ability of erastin analogs to trigger ferroptosis is quantitatively linked to their ability to inhibit system xc− function. Figure 3 Download asset Open asset Structure activity relationship (SAR) analysis of erastin. (A) Structures of 20 erastin analogs. (B) Lethal EC50 for each analog determined in HT-1080 cells in a 10-point, twofold dilution assay, starting at a high concentration of 20 μM, ±β-ME (18 μM). Data represent mean and 95% confidence interval (95% C.I.) from three independent biological replicate experiments. Also reported are IC50 values for inhibition of glutamate release as determined in CCF-STTG1 cells. These data represent the average of two experiments. All values are in μM. ND: not determined. (C) Dose-response curves for selected erastin analogs (3, 14 and 21) in BJeLR and BJeH cells. Data represent mean ± SD from three independent biological replicates. https://doi.org/10.7554/eLife.02523.008 We investigated the above data set in more detail for insights into the erastin structure activity relationship. Erastin's quinazolinone core (Region A) is found in a number of biologically active compounds and is considered to be a ‘privileged’ scaffold (Welsch et al., 2010). Modifications to this region (4–10), including substitution of the quinazolinone for quinolone or using a synthesis (Supplementary file resulted in to of lethal potency compared to suggesting that the quinazolinone core scaffold is essential for the lethality of erastin. Modifications to the (Region B, were not with completely inactive in both the HT-1080 lethality and CCF-STTG1 glutamate release We that of this of the scaffold is essential for activity and that an in the number of in this region results in a higher of lethal to the (Region were in in potency that correlated with inhibition of system xc− to including of the with a of the substituent with a or of it or both lethality and system xc− inhibitory by the potency of and and the of the may a key with the et al., that is essential for Finally, to including of a group a or a substituent resulted in or in lethal potency that were by to in the inhibition of system xc− activity compared to the most potent inhibited glutamate release with 5 potency in the CCF-STTG1 these more potent compounds potently triggered lethality in HT-1080 cells via ferroptosis, as death was suppressed by we have shown that erastin and lethal analogs selective lethality towards human to human and small and compared to cells only et al., 2003; Yang et al., 2014). We tested the most potent lethal analog with the compound (3) and a non-lethal analog in these cell While 14 was we found that both lethal analogs and selectivity towards BJeLR vs BJeH cells (Figure Consistent with the of lethality observed in HT-1080 cells, was a more than more potent lethal molecule compared to 3 EC50 of C.I. vs C.I. these results that it is possible to improve the potency of the erastin scaffold while via to The combination of these new structural with results that the metabolic of erastin (Yang et al., 2014), may in suitable compounds for clinical The effects of erastin on the are to of cystine the above we hypothesized that the effects of erastin were to inhibition of system xc− function and the of cystine from the intracellular with β-ME all effects from erastin To test this hypothesis in a we examined of in the using RNA sequencing of mRNA from HT-1080 cells treated for 5 hr with erastin β-ME (18 µM) or erastin + two independent biological we an average of unique and unique of in both data and of we identified with more and with twofold in vs DMSO-treated controls (Figure Data available as from Dryad data Repository [Dixon et al., In support of the erastin-induced in mRNA expression were by with β-ME for all genes and for each of the These results suggest that the effects of erastin on cellular at the of mRNA expression are to of intracellular cystine, as a of inhibition of system xc− function. Figure 4 with 1 supplement see all Download asset Open asset of erastin effects using (A and B) of genes upregulated (A) and (B) by erastin as in HT-1080 cells using RNA The number of fragments of of was and is as a between the different conditions. expression Data represent the average of two independent biological for each (C and D) mRNA expression of CHAC1 determined by RT-qPCR in HT-1080 and Calu-1 cells in response to erastin ±β-ME treatment for 5 Data are from three independent biological and as mean ± SD and were analyzed by ANOVA with Bonferroni post-tests, ***p<0.001, ns = not significant. In is indicated to the DMSO (E) CHAC1 mRNA levels in different erastin-sensitive cell lines treated with erastin or STS Results in E were analyzed using the ***p<0.001, ns = not significant. Erastin triggers an endoplasmic reticulum (ER) stress We that several of the genes upregulated by erastin were associated with activation of the of the ER stress response et al., et al., Consistent with we observed that the genes were significantly for related to the ER stress and protein response to endoplasmic reticulum stress, activation of protein activity involved in protein positive of activity, The of the ER protein response can be upregulated by amino acid et al., which we is linked to intracellular downstream of system xc− inhibition by erastin. We investigated further the between erastin treatment and activation of the and observed to DMSO-treated erastin treatment (5 μM, resulted in of and of at the protein (Figure supplement 1A). We no for of the which a for activation of a parallel ER stress response (Figure supplement In HT-1080 cells, with the transcriptional inhibitor (1 inhibited erastin-induced in gene expression and but did not erastin-induced cell death (Figure supplement Thus, while of system xc− by erastin other agents, see can trigger a transcriptional of ER stress, it is that this transcriptional response is essential for the lethality observed erastin a pharmacodynamic marker for erastin would be useful to cells are to system xc− inhibition, such as in response to erastin We therefore the profiles for suitable the most gene observed in HT-1080 cells by was CHAC1 Figure an ER gene known to be upregulated downstream of et al., 2006; et al., 2009). We validated these results by RT-qPCR using from HT-1080 and Calu-1 cells, and confirmed that CHAC1 was by with β-ME (Figure CHAC1 mRNA upregulation was observed in response to different active erastin analogs described above (3, and in a recent and et al., low levels of CHAC1 upregulation were also observed in response to two potent analogs both of which some ability to inhibit system xc− function, see Figure suggesting that the induction of ER stress and CHAC1 upregulation may be more sensitive to the inhibition of system xc− than cell viability (Figure supplement 1E,F). We examined the of the above observed transcriptional upregulation of CHAC1 treatment with system xc− inhibitors