Xct-Driven Expression of GPX4 Determines Sensitivity of Breast Cancer Cells to Ferroptosis Inducers

antioxidants

Article xCT-Driven Expression of GPX4 Determines Sensitivity of Breast Cancer Cells to Ferroptosis Inducers

Namgyu Lee 1, Anne E. Carlisle 1, Austin Peppers 1, Sung Jin Park 2, Mihir B. Doshi 1 , Meghan E. Spears 1 and Dohoon Kim 1,*

1 Cell and Cancer Biology, Department of Molecular, University of Massachusetts Medical School, Worcester, MA 01604, USA; [email protected] (N.L.); [email protected] (A.E.C.); [email protected] (A.P.); [email protected] (M.B.D.); [email protected] (M.E.S.) 2 Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01604, USA; [email protected] * Correspondence: [email protected]

Abstract: Inducers of ferroptosis such as the glutathione depleting agent Erastin and the GPX4 inhibitor Rsl-3 are being actively explored as potential therapeutics in various cancers, but the factors that determine their sensitivity are poorly understood. Here, we show that expression levels of both subunits of the cystine/glutamate antiporter xCT determine the expression of GPX4 in breast cancer, and that upregulation of the xCT/selenocysteine biosynthesis/GPX4 production axis paradoxically renders the cancer cells more sensitive to certain types of ferroptotic stimuli. We ﬁnd that GPX4 is strongly upregulated in a subset of breast cancer tissues compared to matched normal samples, and that this is tightly correlated with the increased expression of the xCT subunits SLC7A11 and Citation: Lee, N.; Carlisle, A.E.; SLC3A2. Erastin depletes levels of the antioxidant selenoproteins GPX4 and GPX1 in breast cancer Peppers, A.; Park, S.J.; Doshi, M.B.; cells by inhibiting xCT-dependent extracellular reduction which is required for selenium uptake Spears, M.E.; Kim, D. xCT-Driven Expression of GPX4 Determines and selenocysteine biosynthesis. Unexpectedly, while breast cancer cells are resistant compared to Sensitivity of Breast Cancer Cells to nontransformed cells against oxidative stress inducing drugs, at the same time they are hypersensitive Ferroptosis Inducers. Antioxidants to lipid peroxidation and ferroptosis induced by Erastin or Rsl-3, indicating that they are ‘addicted’ to 2021, 10, 317. https://doi.org/ the xCT/GPX4 axis. Our ﬁndings provide a strategic basis for targeting the anti-ferroptotic machinery 10.3390/antiox10020317 of breast cancer cells depending on their xCT status, which can be further explored.

Academic Editor: Keywords: ferroptosis; GPX4; Erastin; Rsl-3; breast cancer; selenium; lipid peroxidation Sergio Granados-Principal

Received: 20 January 2021 Accepted: 17 February 2021 1. Introduction Published: 20 February 2021 Ferroptosis is a form of cell death that involves an iron-dependent accumulation of lipid peroxides. Several cellular components have emerged as playing key roles in the Publisher’s Note: MDPI stays neutral regulation of ferroptosis. The cystine-glutamate antiporter xCT which is composed of the with regard to jurisdictional claims in subunits SLC7A11 and SLC3A2, plays a protective role against ferroptosis by allowing published maps and institutional afﬁl- iations. the import of cystine, which is a rate-limiting step in the biosynthesis of the antioxidant molecule glutathione [1]. The enzyme glutathione peroxidase 4 (GPX4) utilizes glutathione as a substrate for reduction, and the activity of GPX4 in reducing lipid peroxidation has been shown to be a key step in the prevention of ferroptosis [2]. GPX4 is a selenoprotein, with a selenocysteine residue in its active site, and thus it Copyright: © 2021 by the authors. requires selenocysteine biosynthesis for its expression and activity [3,4]. The selenocysteine Licensee MDPI, Basel, Switzerland. biosynthesis pathway incorporates selenium, such as in the dietary form selenite, to This article is an open access article produce selenocysteinyl-tRNA. The antioxidant function of selenium as a nutrient is distributed under the terms and conditions of the Creative Commons mediated through selenoproteins such glutathione peroxidases and thioredoxins, including Attribution (CC BY) license (https:// GPX4, thus implicating selenium in ferroptosis as well [5,6]. Furthermore, it was found creativecommons.org/licenses/by/ that elevated SLC7A11 expression in cancer cells drives selenocysteine biosynthesis by 4.0/). promoting selenite reduction and uptake as an early step in selenocysteine biosynthesis [4].

Antioxidants 2021, 10, 317. https://doi.org/10.3390/antiox10020317 https://www.mdpi.com/journal/antioxidants Antioxidants 2021, 10, 317 2 of 12

Thus, SLC7A11 allows both the production of glutathione, and the production of the enzyme (GPX4) that utilizes it. Breast cancer, along with lung cancer, is the most common cancer in the world [7]. While successful treatment paradigms have emerged for breast cancers expressing estro- gen, progesterone, or Her2 receptors, triple negative breast cancers (TNBCs) that do not express any of these receptors are refractory to conventional chemotherapeutics and have a markedly poor prognosis [8]. Well-established chemical inducers of ferroptosis, such as the GPX4 inhibitor Rsl-3 and the xCT inhibitor Erastin, and their derivatives, are currently being explored as potential therapeutics for breast cancer therapy and have been shown to exert toxicity in these and other cancer cells [9]. On the other hand, what determines the sensitivity and resistance of cells to these compounds is a critical issue that must be further explored to develop ferroptotic induction as a valid therapeutic strategy for cancer. Here, we examined the expression and functional importance of ferroptotic machinery between transformed and nontransformed cells. We observe that expression of the xCT components SLC7A11 and SLC3A2 are elevated in a subset of patient breast tumor tissues compared to normal breast tissue, and that their coordinated expression tightly correlates with GPX4 expression. We find that the xCT inhibitor Erastin disrupts expression of antioxidant selenoproteins by impairing xCT mediated extracellular reduction, which is required for uptake of selenite and selenocysteine biosynthesis, indicating that modulation of selenoprotein expression is a potentially important and previously unrecognized function of Erastin. In a panel of breast cancer and nontransformed immortalized breast epithelial cell lines, we find the breast cancer lines to be resistant to hydrogen peroxide, consistent with increased GPX4 function. Paradoxically, the breast lines are at the same time hypersensitive to the Erastin and Rsl-3 indicating that breast cancer cells are addicted to the xCT/GPX4 axis to prevent ferroptosis. Our findings indicate a therapeutic window and mechanism for compounds targeting the antiferroptotic machinery of breast cancer cells.

2. Materials and Methods All reagents used in this paper are listed in Supplementary Table S1.

2.1. Cell Lines and Cell Culture ◦ All cell lines were cultured in a humidiﬁed incubator at 37 C under 5% CO2. The cell line information used in this paper is provided in Supplementary Table S2.

2.2. Western Blot Analysis Cells were washed with cold PBS and lysed in RIPA lysis buffer with protease inhibitor (Sigma Aldrich, St. Louis, MO, USA). After 20 min incubation on ice, lysates were centrifuged at 12,000× g at 4 ◦C for 10 min to collect supernatant. Protein concentration of each samples was determined by Bradford assay. Typically, 15–25 µg of total protein were denatured in 6× Lamelli buffer (Boston bioproducts, Boston, MA, USA), loaded per lane on SDS-PAGE gel (Biorad, Hercules, CA, USA), and analyzed by standard immunoblotting. The target proteins were detected by ECL, Pico or diluted Femto substrate (Thermo Fisher Scientiﬁc, Waltham, MA, USA).

2.3. Cell Viability Assay The effect of GPX4 inhibition is affected by cell conﬂuency (31341276). Thus, all viability assays were performed with consistently regarding cell seeding number and time points. For testing toxicity of hydrogen peroxide, Erastin and Rsl-3 in cell lines, 1500 cells were plated in a 96 well plate. Next day, cells were treated with the drugs. Cell viability was measured by CellTiter-Glo Luminescent Assay (Promega, Madison, WI, USA).

2.4. Conditioned Media Thiol Quantiﬁcation (Ellman’s Test) 10,000 cells were plated per well of a 96 well plate. The next day, the media was changed to 100 µL fresh media containing vehicle or Erastin. After 12 h conditioning, the Antioxidants 2021, 10, 317 3 of 12

conditioned media were collected and directly mixed with 50 µL of 10 mM DTNB (5,50- dithiobis-(2-nitrobenzoic acid)) dissolved in DMSO in another 96 well plate. Absorbance at 450 nm was measured spectrophotometrically in 3 min (DTX880, Beckman Coulter, Indianapolis, IN, USA). The blank value was subtracted as noise, and all values were normalized to that of unconditioned media. The leftover cells in the 96 well plate were subjected to the CellTier-Glo Luminescent Assay after adding fresh media. As phenol red mask the changed color of DTNB, phenol red-free media was used for this assay.

2.5. Total Selenium Measurement by ICP-MS Analysis 3 million CAL120 and 4 million MDAMB231 cells were plated in 150 pi dish, and media were changed with that containing vehicle, 12 µM selenite, or 12 µM Erastin the next day. After 2 h of treatment, cells were harvested and washed three times with cold PBS, and weighed. The cell pellets were treated with 500 µM 1:1 H2O2/HNO3 and stood in a safety hood for 12 h at room temperature with periodic venting. Samples were then sonicated for 1 h at 35 kHz and 40 ◦C with periodic venting. Contents of the Eppendorf tubes were then transferred to a glass digestion tube with ASTM type I water (2 × 1 mL). Tubes were sealed, heated at 140 ◦C for 2 h, then cooled and diluted to 5 mL with ASTM type I water for selenium analysis. Selenium measurements were carried out with an Agilent 7500A ICP-MS system which had a standard concentric nebulizer, a Peltier-cooled and double-pass Scott-type spray chamber, torch shield, and standard Nickel interface cones. Between each analytical sample, the probe sample introduction system was rinsed with 10% nitric acid for 60 s to prevent carryover. Calibration curves were made with standard solutions of Se (Ultra Scientific, Kingstown, RI, USA) and QC samples using a multi-element standard (Environmental Express).

2.6. Detection of Lipid Peroxidation Cells were collected by trypsinization and washed with HBSS. The cell pellets were resuspended with 150 µL of 5 µM BODIPY™ 581/591 C11 lipid peroxidation sensor (Thermo Fisher Scientific, Waltham, MA, USA) in HBSS and incubated at 37 ◦C. After 20 min incubation, 500 µL of HBSS was added to the stained cells and subjected to FACS analysis with BD LSR II flow cytometer immediately. Briefly, SSC-A and FSC-A gating strategy was applied to remove the cell debris. FSC-H and FSC-A subgating was performed to identify single cell population. Around 10,000 cells gated as single were used for analysis. BD FACS Diva program and FLOWJO10 were used for data collection and data analysis, respectively.

2.7. RNA Expression and Prognostic Value SLC7A11 and SLC3A2 in Normal and Tumor Tissues Analyses of gene expression data in breast normal and cancer tissues, and disease free survival and overall survival in patients with breast cancer were performed using the web tool GEPIA [10]. Total numbers of samples for each analysis are not designated by the user; instead the GEPIA web tool designates the high and low from the median and provides the total numbers. Total numbers may differ between the different genes (SLC7A11 vs. SLC3A2) due to some samples not meeting the GEPIA criteria for either high or low designation for the given gene.

2.8. Processing of Human Breast Tissues Human breast cancer samples and normal breast tissues were obtained with informed consent from the University of Massachusetts Medical School Biorepository and Tissue Bank using procedures which were conducted under an Institutional Review Board (IRB)- approved protocol. After surgical removal, fresh tumor tissues or normal tissues were immediately snap-frozen in liquid nitrogen and stored at −80 ◦C. Later, tissues were homogenized in RIPA buffer with complete protease inhibitor cocktail, then centrifuged at 13,000× g at 4 ◦C for 10 min. Samples were normalized for protein content and western blots were performed. Protein bands were quantiﬁed using Image J program. Scanned ﬁlms were inverted, and the intensity of each band was measured, and then the background Antioxidants 2021, 10, 317 4 of 12

value was subtracted. The intensity value of protein bands for each sample was normalized to the intensity value for Actin or GAPDH proteins.

2.9. Quantification and Statistical Analysis Results of the viability assay, comparison of protein expressions between normal and cancer tissues, Ellman’s test and total selenium measurement were analyzed using Student’s t test. Disease free survival and overall survival in patients with breast cancer were compared between xCT high and low groups using the Kaplan–Meier method, and significant differences in curves were assessed using the log-rank test. Values of p < 0.05 were considered statistically significant, and data marked with a one (*), two (**) or three (***) asterisks indicate p values of <0.05, <0.01 and <0.001, respectively.

3. Results 3.1. Expression of xCT, an Initial Step of Selenocysteine Synthesis Pathway, Is Highly Upregulated in Breast Cancer Tissues and Expression of GPX4 Is High in xCT Positivie Tumors As xCT activity was shown to drive the expression of the selenoprotein GPX4 via the selenocysteine biosynthesis pathway (Figure1a, [ 4]), we examined the levels of SLC7A11, SLC3A2, and GPX4 in breast normal and cancer tissues to confirm if this pathway is altered in cancer tissues and if GPX4 expression is associated with the expression of xCT. First, we analyzed normalized mRNA transcript levels of SLC7A11 and SLC3A2 from data from The Cancer Genome Atlas (TCGA) compared with normal tissues from (GTEx), using the web tool GEPIA [10], which demonstrated a trend towards increased mRNA levels which was not significant (Figure1b); similar non-significant trends for increase in both subunits was observed in data mining across multiple cancer types (Supplementary Figure S1a). When directly examining protein levels from patient-derived breast tumor samples and normal breast tissues, we found that the expression levels of the xCT subunits, SLC7A11 and SLC3A2, were significantly upregulated in breast tumor tissues compared with normal tissues (Figure1c,d). xCT requires both these subunits [ 4], thus we tried to distinguish between tissues that express only one or both of the subunits. Interestingly, there were 6 cancer tissues out of 14 that expressed significant levels of both SLC7A11 and SLC3A2, which we designated xCT positive tissue (Figure1e), while none of the normal breast tissues was xCT positive (Figure1c). Interestingly, when comparing expression across all tumor samples versus all normal tissue samples, in contrast to SLC3A2 and SLC7A11, GPX4 was not significantly overexpressed in cancer tissues compared to normal tissues across the set (Figure1d). In contrast, when directly comparing the protein expression levels of GPX4 in xCT-positive tumors with their paired (i.e., from the same patient) normal breast tissue, or comparing all xCT positive tumors tissues with all xCT-negative tumor tissues, we saw statistically significant increases of GPX4 expression in the xCT positive group (Figure1f). Furthermore, analyses of TCGA data indicated that high expression of both subunits of xCT was significantly associated with poor overall survival (Figure1g) and disease-free survival (Figure1h) in patients with breast cancer. In contrast, expression of either SLC7A11 or SLC3A2 alone did not have significant prognostic value (Supplementary Figure S2a,b). These findings support the notion that xCT drives the expression of GPX4 [4], and that the elevated expression of both SLC7A11 and SLC3A2 is a determinant for increased xCT function as indicated by GPX4 expression—And is associated with poor prognosis in breast cancer. Antioxidants 2021, 10, 317 5 of 12 Antioxidants 2021, 10, 317 5 of 12

Figure 1. ExpressionExpression of of both both subunits subunits of of xCT xCT correlates correlates with with elevated elevated GPX4 GPX4 expression expression in breast in breast cancer cancer patient patient tumors. tumors. (a) (Schematica) Schematic diagram diagram of the of the selenocysteine selenocysteine synthesis synthesis pathway pathway which which produces produces selenocysteinyl selenocysteinyl tRNA tRNA required required in in selenopro- selenopro- teins suchsuch asas GPX4.GPX4. CreatedCreated withwith BiorenderBiorender ( https://app.biorender.com/(https://app.biorender.com/)).. ( (bb)) Analyses Analyses of of normalized normalized mRNA transcript levels from from tumor tumor (TCGA) (TCGA) and and normal normal breast breast tissue tissue (GTEx) (GTEx) datasets, datasets, conducted conducted using GEPIA using ( http://gepia2.cancer-pku.cn/ GEPIA (http://gepia2.cancer-) webpku.cn/ tool.) web (c) Immunoblots tool. (c) Immunoblots of SLC3A2, of SLC3A2, SLC7A11, SLC7A11, GPX4, and GPX4, Actin and in breastActin normalin breast and normal cancer and tissues. cancer (d tissues.) Comparison (d) Com- of Actin-normalizedparison of Actin-normalized band intensities band ofintensities SLC3A2, SLC7A11,of SLC3A2, and SLC7A11, GPX4 proteins and GPX4 between proteins all breast between normal all breast and cancer normal tissues. and cancer tissues. (e) Dot plot of Actin-normalized band intensities of SLC3A2 and SLC7A11 in all samples shown in c. Sample (e) Dot plot of Actin-normalized band intensities of SLC3A2 and SLC7A11 in all samples shown in c. Sample deﬁned as defined as xCT positive tumor is indicated with asterisks. (f) Comparison of band intensities of GPX4 between xCT posi- xCT positive tumor is indicated with asterisks. (f) Comparison of band intensities of GPX4 between xCT positive tumors tive tumors and their paired normal tissues. The cases of xCT positive tumor and each paired normal tissue are indicated andwith their red and paired blue normal in panel tissues. (c). (g The,h) Expression cases of xCT of positive both SLC3A2 tumor and eachSLC7A11 paired estimates normal tissueoverall are survival indicated (g) withand disease red and- bluefree survival in panel (ch).) for (g,h patients) Expression with of breast both SLC3A2cancer (Median and SLC7A11 cutoff; estimatesLow SLC3A2 overall and survival SLC7A11, (g) and n = disease-free400; High SLC3A2 survival and (h) forSLC7A11, patients n with= 400). breast Data cancer marked (Median with a cutoff;one (*), Low two SLC3A2(**) or three and (***) SLC7A11, asterisksn = indicate 400; High p values SLC3A2 of <0.05, and SLC7A11, <0.01 andn <=0.001. 400). Datans: no marked significance with. a one (*), two (**) or three (***) asterisks indicate p values of <0.05, <0.01 and <0.001. ns: no signiﬁcance.

3.2. Erastin Targets thethe SeleniumSelenium UptakeUptake and and Selenoprotein Selenoprotein Expression Expression Promoting Promoting Activity Activity of xCTof xCT Erastin is an established xCT inhibitor and inducer of ferroptosis, and is currently beingErastin explored is an as established a potential xCT breast inhibitor cancer therapeuticand inducer agent. of ferroptosis, The ferroptosis and is currently inducing beingactivity explored of Erastin as a and potential other xCTbreast inhibitor cancer compoundstherapeutic agent. have beenThe primarilyferroptosis attributed inducing activityto the inhibition of Erastin ofand cystine other importxCT inhibitor leading compounds to glutathione have depletion, been primarily as cysteine attribute whichd to thecan inhibition be formed of from cystine the import reduction leading of cystine to glutathione is a rate-limiting depletion, precursor as cysteine for which glutathione can be formedbiosynthesis from [the11]. reduction The role of cystine xCT in promotingis a rate-limiting selenium precursor uptake for and glutathione selenoprotein biosyn- ex- thesispression [11]. suggested The role to of us xCT that in Erastin promoting may alsoselenium function uptake to disrupt and selenopro the selenium-dependenttein expression suggestedexpression to of us GPX4, that whichErastin given may thealso role function of GPX4 to disrupt in ferroptosis the selenium may greatly-dependent affect aexpres- cancer sioncell’s of sensitivity GPX4, which or resistance given the role to ferroptosis. of GPX4 in xCTferroptosis is thought may togreatly promote affect selenium a cancer entrycell’s sensitivityinto a cell becauseor resistance some to of theferroptosis. imported xCT cystine is thought is intracellularly to promote reduced selenium to cysteineentry into and a cellexported, because providing some of extracellularthe imported reduced cystine thiolis intracellularly groups which reduced allow selenite to cysteine (SeO and3) to ex- be ported,reduced providing to selenide extracellular to initiate the reduced selenocysteine thiol groups biosynthesis which allow pathway selenite (Figure (SeO32)a) to [be4, 12re-]. ducedWe found to selenide that Erastin to initiate treatment the in selenocysteine breast cancer cellsbiosynthesis diminished pathway the levels (Fig ofure extracellular 2a) [4,12]. Wethiols found (Figure that2 b),Erastin eliminated treatment their in selenite breast cancer uptake cells (Figure diminished2c), and reducedthe levels expression of extracellu- of larthe thiols selenoprotein (Figure 2b), antioxidants eliminated GPX1their selenite and GPX4 uptake (Figure (Fig2ured,e), 2c), indicating and reduced that expression Erastin is of the selenoprotein antioxidants GPX1 and GPX4 (Figure 2d,e), indicating that Erastin is Antioxidants 2021,, 10,, 317 317 66 of of 12 12

disruptingdisrupting xCT xCT-mediated-mediated extracellular extracellular reduction reduction which which leads leads to to selenium selenium uptake uptake and and se- se- lenoproteinlenoprotein production inin thesethese cells.cells. TheseThese findings findings support support the the model model for for xCT xCT function function in inGPX4 GPX4 expression expression which which we we previously previously proposed proposed [4]: [4]: that that xCT, xCT, by by allowing allowing cystine cystine import im- portwhich which is in is turn in turn reduced reduced and and exported exported to provide to provide extracellular extracellular thiols, thiols, results results in selenitein sele- nitereduction reduction and and uptake, uptake, leading leading to production to production of selenoproteins of selenoproteins such such as GPX4.as GPX4. Erastin, Erastin, by byinhibiting inhibiting the the cystine cystine import import function function of of xCT, xCT, hinders hinders this this process process and and the the production production of ofGPX4. GPX4. These These doses doses of Erastinof Erastin (3 or(3 6orµ M)6 μM) did did not causenot cause significant significant toxicity toxicity at the at time the pointtime pointfor these for these experiments experiments (Supplementary (Supplementary Figure Fig S3a,b),ure S indicating3a,b), indicating that cellular that cellular toxicity toxicit is noty a isreason not a for reason reduced for extracellularreduced extracellular thiols, selenium thiols, uptake, selenium and uptake, GPX expression. and GPX Furthermore, expression. Furthermore,we observed thatwe observed Erastin did that not Erastin decrease, did andnot decrease, actually slightlyand actually increased, slightly expression increased, of expressionSLC3A2 and of SLC7A11SLC3A2 and subunits SLC7A11 in these subunits cells (Figure in these2d,e), cells supporting (Figure 2d that,e), suppor Erastinting worked that Erastinthrough worked direct inhibitionthrough direct of xCT inhibition activity of rather xCT activity than impairing rather than xCT impairing subunit expression.xCT subu- nitCollectively, expression. these Collectively, findings these further findings support further the role support of xCT the in role driving of xCT the in expression driving the of expressionantiferroptotic of antiferroptotic agent GPX4, andagent suggest GPX4, thatand inhibitionsuggest that of inhibiti the selenocysteineon of the selenocysteine biosynthesis biosynthesisaxis is an important axis is an consequence important consequence of xCT inhibition of xCT by inhibition Erastin. by Erastin.

FigureFigure 2. Erastin downregulates thethe expression expression of of GPX4 GPX4 and and GPX1 GPX1 via via inhibition inhibition of seleniumof selenium uptake. uptake. (a) Schematic(a) Schematic diagram dia- gramof the of proposed the proposed role of role xCT of in thexCT selenoprotein in the selenoprotein synthesis synthesis pathway. pathway. Created withCreated Biorender with Biorender (https://app.biorender.com/ (https://app.bioren-). der.com/).(b) Total thiol (b) quantiﬁcation Total thiol quantification of conditioned of conditioned media from media vehicle from or 6 µ vehicleM Erastin or 6 treated µM Erastin MDAMB231 treated and MDAMB231 CAL120 cells, and CAL120conditioned cells, for conditioned 12 h. Each valuefor 12 wash. Each normalized value was to thatnormalized of unconditioned to that of mediumunconditioned (UCM), medium set to 1. ((UCM),c) Selenium set to uptake 1. (c) Selenium uptake in vehicle or Erastin-treated CAL120 and MDAMB231 cells. Total intracellular selenium levels were in vehicle or Erastin-treated CAL120 and MDAMB231 cells. Total intracellular selenium levels were measured following measured following treatment with 12 µM selenite and/or Erastin for 2 h. (d) Immunoblots of GPX1, GPX4, SLC3A2, treatment with 12 µM selenite and/or Erastin for 2 h. (d) Immunoblots of GPX1, GPX4, SLC3A2, SLC7A11, and Actin in SLC7A11, and Actin in 3 µM, 6 µM Erastin or vehicle-treated cells for 48 h. (e) Quantification of actin-normalized band µ µ intensities3 M, 6 M of Erastin GPX4, orGPX1, vehicle-treated SLC3A2 and cells SLC7A11 for 48 h. relative (e) Quantiﬁcation to the vehicle, of actin-normalizedfrom multiple experiments. band intensities Error ofbars GPX4, represent GPX1, theSLC3A2 mean and ± s.d. SLC7A11 (n = 3). relativeThree independent to the vehicle, experiments from multiple were experiments. performed Error for b bars and representc and each the experiment mean ± s.d. contains (n = 3). Threethree biologicalindependent replicates. experiments The bars were for performed b represent for the b andmean c and± s.d. each of averaged experiment values contains from threeeach experiment. biological replicates. The bars Thefor c bars are thefor mean b represent ± s.d. of the biological mean ± s.d.triplicates of averaged from a values representative from each set. experiment. Data marked The with bars a for three c are (***) the asterisks mean ± indicates.d. of biological p values oftriplicates <0.001. from a representative set. Data marked with a three (***) asterisks indicate p values of <0.001. Antioxidants 2021, 10, 317 7 of 12

Antioxidants 2021, 10, 317 7 of 12

3.3. Breast Cancer Cells Have Increased Resistance against Cell Death Induced by Reactive Oxygen3.3. Breast Species Cancer Which Cells Correlates Have Increased with xCT Resistance Expression against Cell Death Induced by Reactive Oxygen SpeciesThe Which role Correlatesof xCT in withdriving xCT both Expression glutathione production and the expression of gluta- thioneThe-utilizing role of xCTenzymes in driving such bothas GPX4 glutathione suggests production that its expression and the expression may promote of glutathione- survival underutilizing oxidative enzymes stres suchs conditions as GPX4 suggests for breast that cancer its expression cells that have may elevation promote survivalin xCT expres- under sion.oxidative We stressfirst identified conditions two for breast TNBC cancer cell cellslines, that MDAMB231 have elevation and in CAL xCT- expression.120, as having We first in- creasedidentified expression two TNBC of cell both lines, SLC7A11 MDAMB231 and SLC3A2, and CAL-120, while astwo having commonly increased used expression non-trans- of formedboth SLC7A11 mammary and SLC3A2,epithelial while lines, two MCF10A commonly and used MCF12A, non-transformed were found mammary to express epithelial only SLC3A2lines, MCF10A but not and SLC7A11, MCF12A, and were are thus found xCT to- expressnegative only (Fig SLC3A2ure 3a,b). but Next, not SLC7A11,we found that and theare thusbreast xCT-negative cancer lines (Figurewere highly3a,b). Next,resistant we to found death that induced the breast by hydrogen cancer lines peroxide were highly (Fig- ureresistant 3c). Thisto death suggests induced that by x hydrogenCT-positive peroxide breast (Figure cancer3 c). cells This have suggests a selective that xCT-positive advantage againstbreast cancer oxidative cells stress. have a selective advantage against oxidative stress.

Figure 3. TNBC lines MDAMB231 and CAL120 are xCT xCT-positive-positive and have increased resistance against hydrogen peroxide compared toto nontransformed,nontransformed, xCT-negative xCT-negative lines lines MCF10A MCF10A and and MCF12A. MCF12A. (a) ( Immunoblotsa) Immunoblots of the of the xCT xCT subunits subunits SLC3A2 SLC3A2 and SLC7A11and SLC7A11 in TNBC in TNBC cell lines cell lines (labeled (labeled red) andred) nontransformed and nontransformed lines (labeledlines (labeled blue). blue). MCF10A MCF10A and MCF12A and MCF12A nontransformed nontransformed lines are categorized as xCT negative, as they express SLC3A2 but not the SLC7A11 subunits. (b) Quantification lines are categorized as xCT negative, as they express SLC3A2 but not the SLC7A11 subunits. (b) Quantiﬁcation of actin of actin normalized band intensities of SLC3A2 and SLC7A11, relative to vehicle, from multiple experiments. (c) Cell normalized band intensities of SLC3A2 and SLC7A11, relative to vehicle, from multiple experiments. (c) Cell viability of viability of each of these cells upon treatment with 200 μM hydrogen peroxide for 24 h, measured using CellTiterGlo, and µ eachexpressed of these relative cells uponto each treatment line treated with with 200 vehicleM hydrogen (=1.0). Error peroxide bars for represent 24 h, measured the mean using ± s.d. CellTiterGlo, (n = 3). Three and independent expressed relativeexperiments to each were line performed treated with for vehicle b and (=1.0).c and each Error experiment bars represent contain the meanthree ±biologicals.d. (n = replicates. 3). Three independent The bars for experiments b represent werethe mean performed ± s.d. of for averaged b and c and values each from experiment each experiment. contain three The biological bars in c replicates.are the mean The ± barss.d. of for bio b representlogical triplicates the mean from± s.d. a ofrepresentative averaged values set. from each experiment. The bars in c are the mean ± s.d. of biological triplicates from a representative set.

3.4. TNBC Cells Are Paradoxically Hypersensitive to Targeting ofof Anti-FerroptoticAnti-Ferroptotic MachineryMachinery by byErastin Erastin and and Rsl-3 Rsl-3 Our results thus far suggest that xCT positive breast cancer cells—Thosecells—Those expressing high levels ofof bothboth SLC7A11SLC7A11 and and SLC3A2—Are SLC3A2—Are able able to to drive drive GPX4 GPX4 expression expression through through the theselenocysteine selenocysteine biosynthesis biosynthesis pathway, pathway, and alsoand arealso able are toable resist to resist lipid prooxidant-inducedlipid prooxidant-in- duceddeath. death. This is This in line is in with line the with known the known function function of both of xCT both and xCT GPX4 and in GPX4 protecting in protecting against againstferroptosis ferroptosis in various in various cell stress cell states. stress states. We next examined the sensitivity of these cancer and noncancer cells to prolonged exposure to agents targeting the xCT and GPX4 anti-ferroptoticanti-ferroptotic machinery, Erastin and RslRsl-3-3 (Fig (Figureure 44a).a). Surprisingly,Surprisingly, inin contrastcontrast toto theirtheir increasedincreased resistanceresistance againstagainst reactivereactive oxygen species, we found that MDAMB231 and CAL120 cells were hypersensitive to both Erastin and and Rsl Rsl-3-3 relative relative to to the the nontransformed nontransformed lines. lines. We observed We observed that treatment that treatment of Eras- of tin,Erastin, even evenin the in absence the absence of a prooxida of a prooxidantnt insult insultsuch as such hydrogen as hydrogen peroxide, peroxide, induced induced a dra- matica dramatic loss of loss cell of viability cell viability (Figure (Figure 4b,c) and4b,c) significant and signiﬁcant accumulation accumulation of lipid of peroxidation lipid perox- speciesidation (Fig speciesure 4d). (Figure The4 dloss). Theof viability loss of induced viability by induced Erastin by or Erastin Rsl-3 treatment or Rsl-3 treatmentappeared toappeared be caused to beby causedlipid peroxidation by lipid peroxidation and ferroptosis, and ferroptosis, as they were as theyrescued were by rescued the lipid by anti- the lipid antioxidant/ferroptosis inhibitor ferrostatin-1 or α-tocopherol (Figure4e,f). Thus, oxidant/ferroptosis inhibitor ferrostatin-1 or α-tocopherol (Figure 4e,f). Thus, while the while the TNBC cells had an augmented defense system mediated by xCT and GPX4 TNBC cells had an augmented defense system mediated by xCT and GPX4 against oxida- against oxidative stress (Figure3c), they were paradoxically more dependent on these pathways, compared to normal cells, to prevent ferroptosis. This suggests an “addiction Antioxidants 2021, 10, x FOR PEER REVIEW 9 of 13 Antioxidants 2021, 10, 317 8 of 12

tive stress (Figure 3c), they were paradoxically more dependent on these pathways, com- scenario”pared to thatnormal can cells, be further to prevent explored ferroptosi in futures. This studies suggests for optimal an “addiction therapeutic scenario” targeting that in breastcan be cancer. further explored in future studies for optimal therapeutic targeting in breast cancer.

FigureFigure 4. 4.xCT/GPX4 xCT/GPX4 suppression of of ferroptosis ferroptosis is isrequired required in transf in transformed,ormed, xCT-positive xCT-positive breast breast cancer cancer cells but cells not but in notnon- in non-transformedtransformed breast breast cells. cells. (a) Schematic (a) Schematic diagram diagram of a ofknown a known working working mechanism mechanism of Erastin of Erastin and Rsl-3. and Rsl-3.Created Created with Bio- with Biorenderrender (https://app.biorender.com/ (https://app.biorender.com/) (b )(andb, cc)) TheThe relativerelative viabilitiesviabilities of Rsl-3 (b)and) and Erastin Erastin (c (c) )treated treated non-transformed non-transformed breastbreast and and breast breast cancer cancer lines. lines. The The viabilities viabilities were were measured measured at 24at h24 and h and 72 h72 after h after treatment treatment of Rsl-3 of Rsl-3 and and Erastin, Erastin, respectively; respectively; values are relative to vehicle-treated control for each cell line (=1.0) (d) FACS analysis of Bodipy 581/591 C11 lipid values are relative to vehicle-treated control for each cell line (=1.0) (d) FACS analysis of Bodipy 581/591 C11 lipid oxidation oxidation in vehicle and Erastin treated MCF10A and MDAMB231 cells. The Bodipy staining was performed at 72 h after inErastin vehicle treatment. and Erastin Red treated and Blue MCF10A indicate and vehicle MDAMB231 and Erastin cells. treatment The Bodipy conditions staining, respectively. was performed Comp-FITC-A at 72 h aftersubset Erastin was treatment.defined as Red a lipid and BlueROS indicatepositive vehiclecells, which and Erastinis indicated treatment in the conditions, right bar graph. respectively. (e) The Comp-FITC-A viabilities of breast subset cancer was deﬁned lines astreated a lipid ROSwith positiveRsl-3 and/or cells, the which lipophilic is indicated antioxidant/ferroptosis in the right bar graph. inhibitor (e) Theferrostatin-1 viabilities (Fer-1). of breast The cancer viabilities lines were treated meas- with Rsl-3ured and/or at 24 h the after lipophilic treatment antioxidant/ferroptosis of the indicated compounds. inhibitor ( ferrostatin-1f) The viabilities (Fer-1). of TheMDAMB231 viabilities treated were measuredwith Erastin at 24 and/or h after treatmentlipophilic of antioxidants the indicated (ferrostatin compounds. 1, α (-tocopherol).f) The viabilities The ofviabilities MDAMB231 were treatedmeasured with at 72 Erastin h. Three and/or independent lipophilic experiments antioxidants (ferrostatinwere performed 1, α-tocopherol). for b, c, e, and The f viabilitiesand each experiment were measured contain at three 72 h. biological Three independent replicates. experimentsThe bars for b, were c, e, performedand f are the for mean ± s.d. of biological triplicates from a representative set. Data marked with a one (*), two (**) or three (***) asterisks (b,c,e,f) and each experiment contain three biological replicates. The bars for (b,c,e,f) are the mean ± s.d. of biological indicate p values of < 0.05, < 0.01 and < 0.001. triplicates from a representative set. Data marked with a one (*), two (**) or three (***) asterisks indicate p values of <0.05, <0.01 and <0.001. ns: no signiﬁcance.4. Discussion The expression of both SLC7A11 and SLC3A2 subunits can be a marker for xCT func- 4. Discussion tion and selenoprotein production capacity of breast cancer cells. xCT consists of two subunits,The SLC7A11 expression and ofSLC3A2. both SLC7A11 High expression and SLC3A2 of either subunits SLC7A11 can and be SLC3A2 a marker has for been xCT functionpreviously and reported selenoprotein in several production tumor types. capacity SLC7A11 of breast is highly cancer expressed cells. xCTin many consists types of twoof tumors subunits, such SLC7A11 as acute myeloid and SLC3A2. leukemia, High brea expressionst cancer, colorectal of either SLC7A11cancer, hepatocellular and SLC3A2 hascarcinoma, been previously glioma, etc., reported and its in high several expression tumor is types. associated SLC7A11 with poor is highly prognosis expressed of pa- in manytients typeswith cancers of tumors [13,14]. such SLC3A2 as acute is myeloidhighly expressed leukemia, in breast breast cancer cancer, [15] colorectal and osteosar- cancer, hepatocellularcoma [16]. Especially, carcinoma, the expr glioma,ession etc., of SLC7A11 and its high alone expression has been utilized is associated to evaluate with poorthe prognosis of patients with cancers [13,14]. SLC3A2 is highly expressed in breast cancer [15] and osteosarcoma [16]. Especially, the expression of SLC7A11 alone has been utilized to Antioxidants 2021, 10, 317 9 of 12

evaluate the expression of xCT [17–22]. To our knowledge, there has not been an approach to determine the expression of both SLC7A11 and SLC3A2 to evaluate the prognostic value of xCT. However, we previously showed that depletion of any subunits makes xCT nonfunctional [4], which means that confirming the expression of one subunit may not represent the existence of functional xCT in cells. In addition, SLC3A2 also comprises the heavy subunit of the large neutral amino acid transporter (LAT1) with a light subunit protein encoded by the SLC7A5 gene [23] which indicates that SLC3A2 may be expressed as a part of LAT1 complex in some cases. Thus, evaluating one subunit could not be an accurate marker for functional xCT. In this study, the expression of both subunits was evaluated side-by-side to evaluate a level of potentially functional xCT in breast normal and cancer tissues. Some tumors expressed only one subunit and others expressed both subunits which we defined as a xCT high tumor. The significantly elevated expression of GPX4 in xCT-positive tumors versus their paired normal tissues, or versus xCT-negative tumors, supports the notion that tumors expressing high levels of both subunits could be an indicator for the existence of the functional xCT which leads to high selenium uptake and upregulation of selenoproteins. However, this relationship between expression levels of xCT subunits and selenoproteins should be validated with larger sets of paired samples to further clarify the effect of the functional xCT on selenium uptake and its relationship with the expression of various selenoproteins in tumor pathophysiology. Erastin and xCT inhibitors as a strategy to inhibit selenium uptake and GPX4 production in cancer cells. Erastin is currently being explored as a cancer therapy agent, in large part due to its role in inducing ferroptosis. Sulfasalazine and sorafenib are other compounds that have been similarly explored as xCT inhibitors, although they may have wider effects and target profile compared to Erastin [24,25]. Modified compounds based on Erastin with improved stability and/or bioavailability have also been developed [26]. Therefore, fully understanding the mechanism for Erastin and related compounds is of importance in further developing these as a therapeutic strategy. Here we show that Erastin drastically impairs extracellular thiol reduction, selenium uptake, and expression of the selenoprotein GPX4. This finding is significant, as the current model of Erastin mode of action only considers that of impairing glutathione biosynthesis. According to our findings, impaired production of GPX4, a key enzyme implicated in ferroptosis prevention which uses glutathione as substrate, is an additional important mechanism of action for Erastin/xCT inhibitors, one that by itself could have ferroptosis triggering properties even if glutathione production were to somehow be restored. A potential workaround for cancer cells around impaired GPX4 production is that aside from the common dietary selenium compound selenite, there are other independent routes by which selenium could be obtained, in particular recycling of selenocysteine obtained from selenium carriers such as SEPP1, or from selenomethionine, both of which would involve the action of selenocysteine lyase (SCLY) in forming selenide from selenocysteine, prior to its conversion to selenocysteinyl-tRNA. Thus, impairing this route of selenocysteinyl-tRNA formation in conjunction with xCT inhibition could have even more potent effects, and the potential functions of SCLY in cancer and ferroptosis should be explored in future studies. ‘Addiction’ of breast cancer cells to xCT/GPX4 anti-ferroptotic machinery. Conversely to the known effect of xCT inhibitors such as Erastin sensitizing or triggering ferroptosis, the increased expression or activity of xCT is implicated in the increased resistance of cancer cells against insults that can trigger ferroptosis, such as hypoxia or chemotherapy [27–29]. Along similar lines, the role of selenium in producing antioxidant selenoproteins forms the very basis of selenium being considered an antioxidant nutritional supplement [30,31]. Recently, selenocysteine delivered via synthetic peptide was shown to protect against ferroptosis in a stroke model [32]. We also recently showed that CRISPR disruption of selenocysteine biosynthesis machinery (SEPSECS, PSTK) hypersensitizes cancer cells against the lipid prooxidant tert-butyl hydroxide (TBH) [4]. Therefore, it was not surprising that the breast cancer lines MDAMB231 and CAL120, which have elevated expression of both xCT subunits, have increased resistance against hydrogen peroxide compared to Antioxidants 2021, 10, 317 10 of 12

the nontransformed immortalized lines MCF10A and MCF12A, which are xCT-negative (Figure3). What was highly unexpected was that at the same time, MDAMB231 and CAL120 are hypersensitive to the drugs which directly target the machinery that allowed them to resist hydrogen peroxide, namely xCT (Erastin) and GPX4 (Rsl-3). This suggests a scenario in which anti-ferroptotic machinery is upregulated in these cancer cells but at the same time they are ‘addicted’ or highly dependent on this machinery even in the absence of an insult such as hydrogen peroxide. How are why are cancer lines addicted? One possibility involves selenide, which is formed by xCT activity in the process of selenocysteine biosynthesis (which ultimately results in GPX4 production). We have shown that the accumulation of hydrogen selenide gas formed during selenocysteine biosynthesis is toxic to cancer cells by increasing ROS levels [4]. As xCT-positive cancer cell lines are producing more selenide, they may be more dependent on the end product GPX4 to neutralize selenide-induced oxidative stress. As a second possibility, cancer cells might be more detrimental to GPX4 inhibition because they have a higher level of polyunsaturated fatty acids (PUFAs). Basal-like breast cancer cell lines are susceptible to ferroptosis due to the expression of acyl-CoA synthetase long-chain family member 4 (ACSL4) which enriches cellular membranes with long polyunsaturated fatty acids (PUFAs) [33]. Since ACSL4 is expressed higher in breast cancer compared to the adjacent normal tissue [34], and PUFAs are targets of lipid peroxidation, cancer cells might be more susceptible to lipid peroxidation and ferroptosis, thus GPX4 is more essential in cancer cells. As a third possibility, as the xCT-positive cancer cells are expected to have increased glutathione production and GPX4 expression, the resulting increased antioxidant capacity may be accompanied by a reduced selective requirement for other anti-ferroptotic or prosurvival signaling activities, meaning that when this xCT/GPX4-mediated protection is removed, these cancer cells are vulnerable compared to ‘nonaddicted’ cells.

5. Therapeutic Implications and Concluding Thoughts We have shown that cancer cells that overexpress both SLC7A11 and SLC3A2 are the ones that should be considered xCT-positive, and that this pathway, through the selenocysteine biosynthesis pathway mediated production of GPX4, impacts a cancer cell’s sensitivity to various ferroptotic inducers. While future studies should delineate the exact underlying mechanism, our ﬁnding that xCT positive cancer cells are simultaneously resistant to lipid peroxidation insult yet hypersensitive to xCT or GPX4 inhibitors raises important therapeutic implications. There are two classes of ferroptotis inducers: those which directly trigger peroxidation such as hydrogen peroxide, and those that target a cell’s defense against lipid peroxidation. Our results imply that in xCT positive cancers, the latter is the desirable approach. xCT is widely reported to be upregulated in different subtypes of cancers, and has been linked with oncogenic mutations such as Keap1 loss, and associated with clinical parameters such as chemoresistance. Our ﬁndings provide a starting point and rationale for targeting anti-ferroptotic machinery of cancer cells depending on their xCT status, which can be further developed in future studies.

Supplementary Materials: The following are available online at https://www.mdpi.com/2076-392 1/10/2/317/s1, Supplementary Figure S1. Expression proﬁles of SLC7A11 (a) and SLC3A2 (b) in total 33 types of normal and tumor tissue samples. Supplementary Figure S2. Prognostic values of SLC3A2 and SLC7A11 in patients with breast cancer. Supplementary Figure S3. Cell viability is not affected by short-term treatment of Erastin. Supplementary Figure S4. Uncropped image of blots. Supplementary Table S1. A list of materials used in this study. Supplementary Table S2. Information of cell lines and media used in this study. Author Contributions: Conceptualization, N.L., A.E.C., and D.K.; methodology, N.L. and D.K.; validation, N.L.; formal analysis, N.L.; investigation, N.L., A.E.C., A.P., S.J.P., M.B.D., and M.E.S.; resources, D.K.; writing—Original draft preparation, N.L.; writing—Review and editing, N.L., S.J.P., and D.K.; visualization, N.L.; supervision, D.K.; project administration, N.L.; funding acquisition, D.K. All authors have read and agreed to the published version of the manuscript. Antioxidants 2021, 10, 317 11 of 12

Funding: This work was supported by the Suh Kyungbae Foundation (SUHF) Young Investigator Award to D.K. Institutional Review Board Statement: The tissue samples were collected in accordance with ethical approval by the UMass Medical School Institutional Review Board (Docket # H-11731). Informed Consent Statement: Not applicable (determined by Institutional Review Board as “Not Human Research”). Data Availability Statement: All raw data that support the findings of this study are available from the corresponding author upon request. Breast cancer datamining was carried out using GEPIA web tool which utilizes datasets from The Cancer Genome Atlas and GTEx datasets (http: //gepia2.cancer-pku.cn/). Conflicts of Interest: The authors declare no conflict of interest.

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