Reduced Expression of miRNA-27a Modulates Cisplatin ......Predictive Biomarkers and Personalized...

Predictive Biomarkers and Personalized Medicine

Reduced Expression of miRNA-27a Modulates CisplatinResistance in Bladder Cancer by Targeting theCystine/Glutamate Exchanger SLC7A11

Ross M. Drayton1, Ewa Dudziec1,2, Stefan Peter1, Simone Bertz3, Arndt Hartmann3,Helen E. Bryant1, and James WF. Catto1

AbstractPurpose:Resistance to cisplatin-based chemotherapy is amajor obstacle to bladder cancer treatment.We

aimed to identify microRNAs (miRNA) that are dysregulated in cisplatin-resistant disease, ascertain how

these contribute to a drug-resistant phenotype, and how this resistance might be overcome.

Experimental Design: miRNA expression in paired cisplatin-resistant and -sensitive cell lines was

measured. Dysregulated miRNAs were further studied for their ability to mediate resistance. The nature

of the cisplatin-resistant phenotype was established by measurement of cisplatin/DNA adducts and

intracellular glutathione (GSH). CandidatemiRNAswere examined for their ability to (i)mediate resistance

and (ii) alter the expression of a candidate target protein (SLC7A11); direct regulation of SLC7A11 was

confirmed using a luciferase assay. SLC7A11 protein andmRNA, andmiRNA-27awere quantified in patient

tumor material.

Results: A panel of miRNAs were found to be dysregulated in cisplatin-resistant cells. miRNA-27a was

found to target the cystine/glutamate exchanger SLC7A11 and to contribute to cisplatin resistance through

modulation of GSH biosynthesis. In patients, SLC7A11 expression was inversely related to miRNA-27a

expression, and those tumors with high mRNA expression or high membrane staining for SLC7A11

experienced poorer clinical outcomes. Resistant cell lines were resensitized by restoring miRNA-27a

expression or reducing SLC7A11 activity with siRNA or with sulfasalazine.

Conclusion: Our findings indicate that miRNA-27a negatively regulates SLC7A11 in cisplatin-resistant

bladder cancer, and shows promise as a marker for patients likely to benefit from cisplatin-based

chemotherapy. SLC7A11 inhibition with sulfasalazine may be a promising therapeutic approach to the

treatment of cisplatin-resistant disease. Clin Cancer Res; 20(7); 1990–2000. �2014 AACR.

IntroductionAround 7.6 million individuals die from cancer annually

(1). Despite recent advances, the prognosis for advancedtumors remains poor. One of the front-line treatments forbladder cancer is cisplatin-based combination chemother-apy, but the effectiveness of this treatment is severely limited

through the development of cisplatin resistance. Mostpatients with advanced bladder cancer typically showa good initial response to treatment, but ultimately 90%of these patientswill suffer a recurrence of cisplatin-resistantdisease (2). In solid tumors, methods to lower cisplatinconcentration within cells, such as increased drug efflux,reduced influx, or sequestration seem to be amongst thepredominant mechanisms of resistance. The latter may beachieved by a variety of compounds, including glutathione(GSH). This thiol-containing tripeptide is synthesized bynearly all cells; it is a strong electron donor and protectsagainst the harmful effects of various endogenous stressesby quenching reactive hydroxyl-free radicals, other oxygen-centered free radicals, and radical centers onDNAand otherbiomolecules (3). In this way, GSH is also able to protectcells from the cytotoxic effects of various chemotherapeu-tics, including cisplatin (4), and radiotherapy. A rate-lim-iting step in GSH synthesis is the availability of cystine (5),which provides the cysteine moiety of GSH. Cystine importis carried out by the hetrodimeric xc-cystine-glutamatetransporter (6), which is composed of SLC3A2 (also known

Authors' Affiliations: 1Academic Unit of Molecular Oncology and Aca-demic Urology Unit, University of Sheffield, Sheffield, United Kingdom;2Nuffield Department of Surgery, University of Oxford, Oxford, UnitedKingdom; and 3Department of Pathology, University of Erlangen, Germany

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

H.E Bryant and J.W.F. Catto shared senior authorship of this work.

Corresponding Authors: James WF. Catto, Academic Unit of MolecularOncology, G Floor, The Medical School, University of Sheffield, Beech HillRoad, Sheffield, S10 2RX,United Kingdom. Phone: 44-114-226-1229; Fax:44-114-271-2268; E-mail: [email protected]; and Helen E. Bryant,E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-2805

�2014 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 20(7) April 1, 20141990

on August 22, 2021. © 2014 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst February 10, 2014; DOI: 10.1158/1078-0432.CCR-13-2805

http://clincancerres.aacrjournals.org/

as 4F2HC) and SLC7A11 (also known as xCT). Upregula-tion of SLC7A11 has been reported as a mechanism ofcisplatin resistance in ovarian cancer (7).Resistance to cisplatin treatment in bladder cancer is

common, and can be mediated through one or more of alarge number of pathways (8). The changes in proteinexpression that underpin these pathwaysmay arise throughgenetic or epigenetic means (8). The latter include changesin DNA methylation and microRNA (miRNA) expression(9). These short single-stranded RNAs play key roles inmany carcinogenic processes (10) and primarily act asnegative regulators of genes through the interaction withthe 30 untranslated region (UTR) of target mRNAs resultingin either mRNA destruction or inhibition of translation(11). Alterations in expression of particular miRNAs fol-lowing the development of cisplatin resistance with func-tional consequences for target mRNAs is well documentedin various cancer cell lines including those derived frombreast (12) and ovarian cancers (13). Here, we presentevidence that a change in expression of miRNA-27a con-tributes to cisplatin resistance in bladder cancer throughmodulating the expression of the SLC7A11 and as a result,increasing levels of intracellular GSH. We demonstrate away to reverse resistance that has clinical potential andsuggest that the biochemical consequences of this mecha-nism of resistance may be used to stratify drug choice inpatients with advanced cancer.

Materials and MethodsCell lines, single cell clones, and cisplatin-resistantvariantsCisplatin-resistant cell lines were generated from hetero-

geneous cells and sensitive single cell clonesusingEJ/T24andRT112 bladder cancer lines. Cells were grown in increasing

drug concentrations (10%–20% increase per passage at 70%confluence) for 4months. Primary cellswere purchased fromAmerican Type Culture Collection and grown in Dulbecco’sModified Eagle Medium with 10% fetal calf serum. Theadapted cell lines are deposited at the Health ProtectionAgency. A2780-DDP cells were obtained from Sigma.

Clonogenic survival assayFive hundred to 2,500 cells were plated in triplicate onto

100-mm dishes 4 hours before the addition of increasingdoses of cisplatin as indicated. Ten days later, when coloniescould be observed, they were fixed and stained with meth-ylene blue in ethanol (4 g/L). Colonies consisting of morethan 50 cells were subsequently counted. Each colony wasassumed to represent one cell surviving from the originalnumber plated and the surviving fraction for each dosecalculated compared with untreated control. When siRNA-depleted cells were used, they were transfected as above for48 hours, then replated in the presence or absence ofincreasing doses of cisplatin.

Quantification of miRNA expressionTotal RNAwas extractedusing themirVanaKit (Ambion).

The expression of 357miRNAs (miR)was determined usingreal-time PCR (RT-PCR) with microfluidic cards (HumanmiRNA v1.0, Applied Biosystems) used as per the manu-facturer’s instructions and analyzed on an ABI 7900HT RT-PCR system.Relative expressionwas calculated from theDCt

value for each miRNA, calculated by the subtraction of theplate mean Ct value from the Ct value for each specificmiRNA. DDCt values between resistant and sensitive lineswere calculated, and used to generate fold changes inexpression using the expression 2�DDCt.

Standalone TaqMan quantitative PCR (qPCR) assays(Applied Biosystems), used according to themanufacturer’sinstructions were also used to confirm downregulation ofmiRNA-27a in resistant cells.

Measurement of cisplatin adducts in DNACells were treated with cisplatin for 2 hours, harvested at

relevant time points, and frozen at �80�C until analysis.Genomic DNA was extracted using a DNeasy Kit (Qiagen),treated with RNAse A (Sigma) to remove cisplatin-damagedRNA. Exactly 1 mg DNA from each sample was loaded intoa dot blotter (Bio-Rad), washed thoroughly with 20�saline-sodium phosphate-EDTA buffer, and baked (80�C,30 minutes). The DNA was then blocked with 5% milk,probed using R-C18 monoclonal antibody (Oncolyse)raised against 1,2 (GG) intrastrand adducts (14), and thenwith horseradish peroxidase -conjugated anti-rat immuno-globulin G (#7707– NEB). Dots were developed using achemiluminescent substrate (GE Healthcare), visualizedand quantified using a LAS-3000 imager (Fujifilm).

Expression of cisplatin transport and sequestrationmolecules

The expressionofmRNAs important for cisplatin transport(ATP7A, ATP7B, SLC31A1; CTR1) and GSH biosynthesis

Translational RelevanceCisplatin-based chemotherapy is the first-line system-

ic treatment for advanced bladder and ovarian cancers.Most patients with these cancers eventually suffer recur-rence with cisplatin-resistant disease. Mechanisms bywhich resistance is achieved are varied and better under-standing of these is essential for the development ofresensitization strategies. We found consistent dysregu-lation of a panel of microRNAs (miRNA) during thedevelopment of cisplatin resistance in bladder cancer,and showed that one of these miRNAs (miRNA-27a)targets a cystine/glutamate exchanger, which mediatesglutathione synthesis SLC7A11. Cancer samples frompatients showed that dysregulation of these moleculesstratified clinical outcomes in patients treated with cis-platin. Cisplatin resistance in our cells could be reversedusing siRNA to the target genes and sulfasalazine, a U.S.Food and Drug Administration–approved drug knownto inhibit SLC7A11.

Cisplatin Resistance in Bladder Cancer

www.aacrjournals.org Clin Cancer Res; 20(7) April 1, 2014 1991




[SLC3A2 (4F2HC), GCLC, GCLM, GSS, GSR, SLC7A11(xCT)] was measured using RT-PCR of cDNA generated byreverse transcription of cellular RNA using random hexam-ers. Primers were validated usingmelting curve analysis, andby inspectionof anelectrophoretic gel for a singlebandof theexpected size. mRNA quantification was normalized to theaverage of b-actin and U1 RNA expression and fold changesin resistant cells were calculated using from DDCt values. Theexpression of ATP7A, SLC31A1, and SLC7A11 proteins weremeasured using Western blotting using primary antibodies(SC-32900 and SC-66847; Santa Cruz Biotechnology) andNB300–318 (Novus).

Cellular glutathione concentrationLevels of intracellular GSH were measured using a GSH-

Glo glutathione assay kit (Promega). In triplicate, 5,000cells were plated, left for 4 hours before measuring theluminescence with a SpectraMax luminometer (MolecularDevices), according to the manufacturer’s instructions. Tris(2-carboxyethyl)phosphine (Thermo) was added to reduceoxidized GSH and ascertain the levels of total GSH presentin each sample.

Restoration of underexpressed miRNA expressionTo modulate miR expression, cells were transfected with

pre-miR miRNA precursors or relevant scrambled negativecontrol RNAs (Applied Biosystems) using LipofectamineRNAiMAX (Invitrogen). Six-well plates were loaded with5 pMol pre-miR in 500 mL serum-free medium to which5 mL RNAiMAXwas added 20minutes before 100,000 cells.Cellswere used after 48 to 72hours anddesired alteration ofmiR expression levelwas confirmedusing aTaqManmiRNAqPCR (Applied Biosystems).

Luciferase reporter assayThe first 1,100 bp of the SLC7A11 30UTR containing the

miRNA-27a–binding site was cloned from EJ-R/EJ mixedcDNA and ligated into pMIR REPORT (Invitrogen). Theresulting pMIR REPORT þ SLC7A11 30UTR plasmid wassubjected to a round of site-directed mutagenesis to intro-duce twomutations into themiRNA-27a–binding site.Dualluciferase assays were conducted in a 6-well plate format,pMIR REPORT þ SLC7A11 30UTR and pMIR REPORT þMutated SLC7A1130UTR (100ng)was transfected into 70%confluent HEK293 cells, along with miScript miRNA-27ainhibitor, or scrambled RNA control (50 pmol/L; AppliedBiosystems) and pRL-TK Renilla luciferase plasmid (0.7 ng).Forty-eight hours posttransfection, firefly and Renilla lucif-erase were quantified sequentially using theDual LuciferaseAssayKit (Promega), and luminescencewasmeasuredusingthe manufacturer’s recommended luminometer (PromegaGlomax). Firefly luciferase expression was quantified andnormalized to Renilla luciferase expression.

siRNA knockdown and small-molecule inhibition ofSLC7A11

SLC7A11 was knocked down using a commercially avail-able ON-TARGET smartpool RNA from Thermo Fisher.

Sulfasalazine was obtained from Sigma. For clonogenicassays, cells were pretreated for 48 hours with siRNA/sulfa-salazine before replating and treatment with cisplatin.

Patients and tumorsTo evaluate cell culture findings in human bladder

cancer, we examined the tumors from 354 patients intwo separate cohorts (Supplementary Tables S1 and S2).The first cohort included 139 primary tumors represent-ing all disease phenotypes, collected prospectively fromthe Royal Hallamshire Hospital, (Sheffield, United King-dom). We microdissected sections of fresh-frozen tissueto enrich for tumor cells (>90%) and extracted total RNA,as above. The second cohort included 215 patients treatedwith adjuvant chemotherapy for invasive bladder cancer.Of these, the majority (n ¼ 149) were from a phase III,multicentre randomized controlled trial conducted by theGerman Arbeitsgemeinschaft Urologische Onkologie(AUO) comparing two adjuvant regimens (cisplatin/methotrexate vs. methotrexate/vinblastine/adriamycin/cisplatin; AUO-AB 05/95; ref. 15). For each patient, weobtained archived formalin-fixed paraffin-embedded(FFPE) tissue and extracted 3 � 1.5 mm cores (33–31AP/25, Miltex Inc) from cancerous regions to construct atissue microarray (TMA) as detailed elsewhere (16, 17).TMA content and construction were controlled by acertified uropathologist (A. Hartmann).

Tissue arrayProtein expression was determined using automated

immunohistochemistry (program: SCC1, Benchmark XT,Ventana Medical Systems, Oro Valley, AZ). For SLC7A11,we used a polyclonal antibody (xCT, Thermo-Scientific;Cat.-No. PA1–16893) at 1:400 dilution for 30 minutes.Sections were visualized with diaminobenzidine chromo-gen and counterstained in a modified Mayer hematoxylin.Negative controls were performed using normal serumrather than SLC7A11 antibody. Membranous staining wasscored for intensity and percentage of positive cells. HighSLC7A11 expression was defined as strong intensity in 15%or more of cells.

Statistical analysesAssociations between miR expression and cisplatin sen-

sitivity were tested using fold changes in resistant andsensitive cells. We identified miRs that consistently showedmore than a 2-fold difference (either gain or loss) in expres-sion between resistant and sensitive cells, and selected thosein which this reached statistical significance (defined as P <0.05, Student t test). We compared these with miR expres-sion profiles in primary bladder cancer (18) generated usingthe same array. Hierarchical clustering was performed usingCluster 3.0 and visualized in Tree view (Eisen Lab).MiRNAsthatwere differentially expressed in resistant comparedwithsensitive cells were used to hierarchically cluster samplesaccording to the cell line and presence/absence of chemore-sistance. Correlation between continuous variables wasassessed using Pearson correlation coefficient within SPSS.

Drayton et al.

Clin Cancer Res; 20(7) April 1, 2014 Clinical Cancer Research1992




For patient outcomes, we compared RNA and proteinexpression with tumor pathology and subsequent pheno-type. Associations between clinicopathological features andtumor behavior were examined using univariable and step-wise multivariable Cox proportional hazards regressionmodels (SPSS Vsn. 14.0; SPSS Inc.). Outcomes with respectto time were plotted using the Kaplan–Meier method andcompared using a log-rank test within SPSS (Vsn. 19.0 SPSSInc.). Tumor progression was defined as the presence ofpathologic, radiologic, or clinical evidence of an increase intumor stage and measured from the time of surgery to thetime of proven event. Where cisplatin sensitivity was com-pared between cell lines or between control cells and cellstreated with a drug, precursor miRNA or siRNA statisticalsignificance was calculated using Student two-tailed t test.

ResultsmiRNA expression is altered in cisplatin-resistantbladder cancer cells

To identify changes inmiRNA expression associated withcisplatin resistance, we compared matched cisplatin-sensi-tive and -resistant bladder cancer cell lines. The latter werederived by culturing the bladder cancer cell line EJ, or singlecell clones derived from the original heterogeneous EJ(namely clones D4 and G7) in media augmented withincreasing cisplatin concentrations for several months. Thisresulted in three cisplatin-resistant cell lines (EJ-R, D4-R,and G7-R), all of which were able to tolerate much higherconcentrations of cisplatin than any of the parental lines(Fig. 1A). IC50 concentrations for cisplatin were typically<1 mmol/L for sensitive lines and 10 mmol/L for resistant

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Figure 1. Characterization of cisplatin-resistant cell lines and miRNA expression. A, clonogenic survival assays of cisplatin-treated bladder cancer cell linesand their cisplatin-resistant derivatives (designated as respective cell line followed by R). Error bars, SD; n ¼ 4. B, fold-change in expression of C.160 miRNAs in three paired sensitive and resistant lines. C, hierarchical clustering using 32 selected miRs (red, low expression; green, high expression)stratifies cells according to cisplatin resistance (orange, resistant; white, sensitive) regardless of cell line or method of derivation.






lines. MiRNA expression profiling in these paired cell linesrevealed that although the expression of the majority ofmiRNAs remained unchanged (Fig. 1B), amiRNA signaturecould be identified that stratified cisplatin resistance (Fig.1C). A second set of paired cisplatin-sensitive and -resistantbladder cancer cell lines, generated in the same way usingRT112 cells as the parental line (RT112 and RT112-R), werealso included in this analysis, and displayed a similarmiRNA profile (Fig. 1C).

Cisplatin induces fewer 1,2 (G,G) intrastrand adductsin resistant cells

Resistance to cisplatin can occur either before DNAadduct formation via changes in cellular metabolismof cisplatin or postadduct formation via changes in DNAdamage repair. To initially characterize the mechanism bywhich cisplatin resistance had arisen in our cells, wemeasured cisplatin-induced 1,2 (G,G) intrastrand DNAcross-links using monoclonal antibodies directed againstthese adducts in EJ and EJ-R cells (14). Two hours fol-lowing treatment-resistant cells displayed significantlyfewer 1,2 (G,G) intrastrand cross-links compared withsensitive cells (Fig. 2A; P < 0.001 for all concentrations ofcisplatin above 1 mmol/L). When cisplatin was removedfrom growth media following a 2-hour treatment of 12mmol/L, in both the sensitive and resistant cell linesadducts were repaired, as evidenced by the reductionin the number of adducts in genomic DNA over thefollowing 48 hours (Fig. 2B and C). The rate at whichthese adducts were removed seemed broadly similarbetween resistant and sensitive lines. This suggests aresistance mechanism related to cisplatin influx/efflux or

to cytoplasmic detoxification of cisplatin before the cyto-toxic interaction with DNA in the nucleus, rather than adifference in ability of resistant cells to tolerate or repaircisplatin-induced DNA damage.

Cisplatin resistance is mediated through increasedexpression of SLC7A11 and increased production ofglutathione

The expression of copper transporter proteins are knownto be responsible for the efflux (ATP7A/ATP7B; ref. 19) andinflux (CTR1; ref. 20) of cisplatin, and their dysregulationhas been linked with cisplatin resistance in some cancers,most notably ovarian (21). Initial analysis of mRNA levelsof these copper transporters revealed that ATP7BmRNAwassignificantly downregulated by approximately 50% in thethree resistant lines, whereas ATP7AmRNAwas significant-ly upregulated by around 40% (Fig. 3A). Increased mRNAexpression of the cisplatin efflux protein ATP7A was notconsidered a likely candidate for the modulation of cisplat-in resistance; however, as this increased mRNA expressionwas not accompanied by an increase in protein levels asevidenced by Western blot analysis (Fig. 3D). CTR1 mRNAwas typically 3-fold upregulated in all resistant cell lineswhen comparedwith corresponding sensitive cell lines (Fig.3A). This is the opposite of what would be expected werethese influx and efflux proteins to be involved in theregulation of cisplatin resistance and, like ATP7A, thisaltered mRNA expression was not reflected in levels of theCTR1 protein (Fig. 3D). This suggests that changes inthe cellular import and export of cisplatin are unlikely tobe responsible for the resistance of our bladder cancer celllines to cisplatin.

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Figure 2. Genotoxicty of cisplatin and measurement of cisplatin/DNA adducts in sensitive and resistant cells. A, measurement of 1,2 (G,G) intrastrandcisplatin-DNA adducts in genomic DNA of EJ and EJ-R cells following a 2-hour treatment with a range of cisplatin concentrations. Error bars, SD;n ¼ 3. P < 0.001 for all concentrations of cisplatin above 1 mmol/L. B, densitromic plot of extent of 1,2 (G,G) intrastrand cisplatin-DNA adductrepair in EJ/EJ-R for 48 hours following a 2-hour treatment with 12 mmol/L cisplatin. Error bars, SD; n ¼ 3. C, example dot blot used to generatedata visualized in Fig. 3B.

Drayton et al.





Following import into cells, cisplatin is able to formconjugates with GSH leading to its sequestration and detox-ification (22), and increasedGSH levels have been observedin other cisplatin-resistant cell lines (23). We found signif-icantly increased concentrations of both reduced (GSH)and oxidized glutathione (GSSG)GSH in cisplatin-resistantcells (Fig. 3B). Resistant cells displayed levels of GSH andGSSG approximately double that of the sensitive parentalline, suggesting that increased GSH production might beresponsible for cisplatin resistance. We examined mRNAexpression of the six major genes whose protein productsare concerned with GSH biogenesis in paired sensitive andresistant cell lines (Fig. 3C).Many of these genes displayed adegree of small but significant upregulation in resistantcells. However, one gene, SLC7A11, was particularly signif-icantly overexpressed in resistant cells, displaying a 10-foldincrease in expression in resistant lines and a high degree ofstatistical significance. We also examined the expression ofSLC7A11 protein and found it to be overexpressed in

resistant cells (Fig. 3D). SLC7A11 is one part of the hetero-dimeric xc-cystine/glutamate exchanger, and as cystine bio-availability is a critical factor in the rate of GSH production,itmay represent thepoint atwhich the increased rate ofGSHproduction in resistant cells is regulated. This suggests thatchanges in the expression of genes involved in GSH pro-duction, particularly SLC7A11, might be responsible forresistance in our resistant cell lines.

Overexpression of miRNA-27a reduces levels ofSLC7A11 and intracellular glutathione, andresensitizes resistant cells to cisplatin

To determine whether SLC7A11 was a candidate forregulation by miRNA, we examined the 30UTR for sites ofpotential miRNA interaction using TargetScan (www.tar-getscan.org) and compared them with our expression pro-files. Of the miRNAs predicted to target the 30UTR ofSLC7A11, we observed that miRNAs-25 and -27a weresignificantly downregulated in our resistant lines and had

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Figure 3. Analysis of candidate pathways mediating cisplatin resistance in sensitive and resistant cells. A, change in expression of three mRNAsconcerned with cisplatin import (CTR1) and export (ATP7A, ATP7B) in three paired cisplatin-sensitive (EJ, D4, G7) and -resistant (EJ-R, D4-R, G7-R)cell lines. B, measurement of intracellular GSH (black bars, reduced GSH; white bars, total GSH and GSSG) in EJ bladder cancer cells and theircisplatin-resistant derivative line EJ. Error bars, SD; n ¼ 3; �, P < 0.01; ��, P < 0.0005. C, change in expression of six mRNAs concerned withGSH biosynthesis in three paired cisplatin-sensitive (EJ, D4, G7) and -resistant (EJ-R, D4-R, G7-R) cell lines. D, Western blot analysis of proteinsconcerned with cisplatin import and export (ATP7A (163 kDa), CTR1 (35 kDa), SLC7A11 (55 kDa) against a-tubulin (55 kDa)) in three paired cisplatin-sensitive(EJ, D4, G7) and -resistant (EJ-R, D4-R, G7-R) cell lines.






appeared in our panel of dysregulated miRs (Fig. 1C), inkeeping with consequential upregulation of SLC7A11.MiRNA-32 was also shown to have the potential to interactwith the 30UTR of SLC7A11, and although it did not appearin our signature of dysregulatedmiRs, it did show consistentdownregulation in all resistant cells compared with theirsensitive parental lines. However, transfection of resistantcells with a commercially available pre-miR precursor tomiRNA-27a, but not miRNA-25 or -32, lowered the expres-sion of SLC7A11 (Fig. 4A) and increased sensitivity by 100-fold following a12mmol/L treatmentwith cisplatin (Fig. 4B).This suggests that reduced expression of miRNA-27a caninduce cisplatin resistance in bladder cancer cells, by reduc-ing the level of cellular SLC7A11. Downregulation of

miRNA-27a in EJ-R cells was also further confirmed bystandalone qPCR (Supplementary Fig. S1). The restorationof cisplatin sensitivity following increased expression ofmiRNA-27a was associated with an increase in 1,2 (G,G)intrastrand adducts following cisplatin treatment (Fig. 4C)and a decrease in intracellular GSH (Fig. 4D), supportingthe hypothesis that miRNA-27a downregulation protectscells from the cytotoxic effect of cisplatin via alterations inGSH biosynthesis. To determine whether the SLC7A1130UTR can be a direct target of miRNA-27a in living cells,we constructed a luciferase reporter assay using the wild-typeand amutant SLC7A11miR-27 seed sequence. Inhibition ofmiRNA-27a production in HEK293 cells using a commer-cially available inhibitory RNA significantly increased

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Figure 4. Effect of miRNA-27a manipulation on SLC7A11 expression, cisplatin sensitivity, and cisplatin genotoxicity in resistant bladder and ovariancancer cells. A, Western blot analysis of SLC7A11 expression following transfection of cisplatin-resistant cells with miRNA-27a, miRNA-25, ormiRNA-32, (mIR-27a, -25, -32). B, effect of increased miRNA-27a expression on cisplatin sensitivity of EJ-R cells compared with a scrambled controloligo as measured by clonogenic assay. Error bars, SD; n ¼ 3. C, effect of increased miRNA-27a expression of level of cisplatin-induced DNA damagein EJ-R cells compared with untreated and scrambled RNA controls. Error bars, SD; n ¼ 3. D, effect of increased miRNA-27a expression on levelsof reduced (black bars) and total (white bars) GSH. �, ¼ P < 0.01. Error bars, SD; n ¼ 3. E, effect on luciferase expression of a miRNA-27a inhibitorin HEK293 cells compared with scrambled control. The luciferase gene was augmented with a portion of the 30 UTR of SLC7A11 containing themiRNA-27a–binding site, or a similar construct with the miRNA-27a abolished by site-directed mutagenesis (mutant). F, effect of increased miRNA-27aexpression (miR-27a) or transfection with a scrambled control (Scrm) on cisplatin sensitivity, the cisplatin resistant ovarian cancer cell line A2780-cDDP asmeasured by clonogenic assay. Error bars, SD; n ¼ 3.

Drayton et al.





expression of luciferase reporter augmentedwith the relevantpart of the SLC7A11 30UTR. This effect was abolished bymutation of the miRNA-27a seed sequence (Fig. 4E).

The cisplatin sensitivity of a resistant ovarian cancercell line can also be increased by modulation ofmiRNA27a levelsCisplatin resistance though SLC7A11 was first reported

when thedrug-resistantovariancancer lineA2780-cDDPwasfound to have higher cystine uptake and intracellular GSHconcentrations than its cisplatin-sensitive parent (7). Resis-tance could be induced with transfection of components ofthe Xc

� transporter (SLC7A11 and SLC3A2). We transfectedA2780-cDDP with pre-miR-27a, and similar to EJ-R cells,transfection resulted in a 10-fold increase in cisplatin sensi-tivity, suggesting that altered levels of miRNA-27a can effectcisplatin sensitivity in other cancer types.

SLC7A11 is a key modulator of cisplatin resistance inbladder cancer cellsEvidence of the importance of SLC7A11 in the regulation

of cisplatin resistance is demonstrated by the increasedsensitivity of formerly resistant EJ-R cells to cisplatin whentransfected with siRNA against SLC7A11 (Fig. 5A and B).Taken together, these data suggest that direct targeting of theSLC7A11mRNA 30UTR bymiRNA-27a, alters GSH levels incells, which can then sequester and detoxify cisplatin,resulting in decreased cisplatin-induced DNA adduct for-mation and consequently cellular resistance to cisplatin.

Inhibition of SLC7A11 with sulfasalazine, at clinicallyrelevant doses, resensitizes cisplatin-resistant bladdercancer cellsThe xc� transporter has recently been implicated as a

causative mechanism for seizures in patients with glioma

(24). Specifically, upregulation of Xc� led to high peritu-

moral concentrations of glutamate, a neuro-excitatory ami-no acid that precipitated seizures (25). Although the role ofXc

� is unknown in glioma, seizures could be reduced in amodel using the proven Xc inhibitor sulfasalazine (26) atdoses equivalent to those used to treat Crohn inflammatorybowel disease (250 mmol/L) in humans (24). We evaluatedthe potential for sulfasalazine to induce cisplatin sensitivityin our cells through Xc- inhibition. We found that this doseof sulfasalazine increased the cisplatin sensitivity of resis-tant cells more than 10-fold compared with a dimethylsulfoxide control (Fig. 5C).

miRNA-27a/b expression correlates with increasedSLC7A11 expression and poor prognosis in patientswith bladder cancer

To examine the clinical significance of our current find-ings, we studied tumors from two distinct patient cohorts.The first included 139 primary tumors representing thedisease spectrum. Although our in vitro data implicatemiRNA-27a in cisplatin resistance, its similarity tomiRNA-27b and the fact that both were downregulated inresistant cells, combinedwith our previous observation thatboth are abnormally expressed in primary bladder cancer(18), lead us to examine the expression of both in this firstcohort. We found that the expression of miRNAs-27a/27bwas directly correlated (r ¼ 0.75; P > 0.001) and inverselyrelated to SLC7A11 mRNA expression (r ¼ �0.64; P >0.001; Fig. 6A and Supplementary Table S1). When out-come was analyzed, tumors with high SLC7A11 and/or lowmiRNA-27a/27b expression progressed more frequently toadvanced disease following treatment, compared withothers (log-rank P < 0.04; Fig. 6B). The second cohortincluded the tumors from 215 patients treated with che-motherapy as part of a randomized control trial comparing

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Figure 5. Effect of SLC7A11 knockdown and inhibition on cisplatin sensitivity. A, use of siRNA directed against SLC7A11 to reduce SLC7A11 protein levelsin EJ-R cells 48 hours posttransfection. B, effect of SLC7A11 knockdown compared with scrambled RNA control on cisplatin sensitivity of EJ-Rcells as measured by clonogenic assay. Error bars, SD; n ¼ 3. ��, P < 0.005. C, effect of sulfasalazine pretreatment on cisplatin sensitivity of EJ-Rcells as measured by clonogenic assay. Error bars, SD; n ¼ 3. �, P < 0.1.






two cisplatin-based regimens (15) with gemcitabine–cis-platin. High membranous expression of SLC7A11 wasidentified in 22 of the 215 (10.2%) tumors and was asso-ciated with poor cisplatin response (P < 0.04; Fig. 6C). Inparticular, patients whose tumors had high SLC7A11 pro-tein expression responded to cisplatin less frequently andfor a shorter duration than thosewith little or no expression.In multivariable analysis, SLC7A11 expression [HR ¼ 2.0;95% confidence interval (CI), 1.1–3.5; P ¼ 0.017] andextent of lymph node involvement (HR ¼ 4.4; 95% CI,1.1–18.5; P ¼ 0.04) were the only variables independentlypredicting progression and cancer-specific survival (Fig. 6Dand Supplementary Table S2).

DiscussionMany previous studies have examined alterations in

miRNA expression during the development of cisplatinresistance in various cancer lines including those derivedfrom hepatocellular carcinoma (27), ovarian cancer (28),non–small cell lung cancer (29), and germline tumors (29).None of these studies identified miRNA-27a as a potentialmarker for or mediator of cisplatin resistance. The action ofspecific miRNAs tends to be highly specific to particulartissues, and consistent with this miRNA-27a has beenreported as having both oncogenic and tumor-suppressiveroles in various tumors. In cancer types such as breast (30)and prostate (31), miRNA-27a has been considered anonco-miR, with high levels of expression being associatedwith a poor prognosis, whereas in acute leukemia, it hasbeen suggested that miRNA-27a functions as a tumor sup-

pressor (32). Consistent with our data suggesting miRNA-27a as a keymediator of drug resistance andprognosis in thecontext of bladder cancer, a recent paper foundmiRNA-27ato be consistently downregulated in FFPE bladder tumormaterial frompatients who experienced disease progressionwhen compared with material from those patients whoexperienced a complete response (33).

MiRNA-27a forms part of a larger signature of miRNAexpression that stratifies cisplatin resistance in bladdercancer. Although many of these miRNAs no doubt havefunctional roles in the mediation of cisplatin resistance,we have uncovered a critical role for miRNA-27a, namelyits decreased expression in cisplatin-resistant bladdercancer modulating SLC7A11 upregulation and therefore,allowing for increased cystine import and increased GSHsynthesis. This linked our miRNA signature with well-established and widely studied mechanisms of cisplatinresistance (34).

When designing our experiments, we were interested inunderstanding whether our mechanism of drug resistancewould arise de novo with carcinogenesis (i.e., in cells notexposed to cisplatin) or be acquired during cisplatin treat-ment. It is likely that both occur in cancer, as many hall-marks of carcinogenesis produce drug resistance as a sec-ondary phenotype (e.g., apoptosis avoidance). In anattempt to model these different scenarios, we deriveddrug-resistant cells from two different cell populations,heterogeneous cells and expansions of single cell clones.Although resistance in formermay reflect the selection of denovo resistant-cells, the latter models acquired resistance bythe conversion of sensitive to resistant cells. It was

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Figure 6. Clinical significance of decreased miR-27a/b expression and increased SLC7A11 expression in bladder tumors. A, expression of miR-27a/27band SLC7A11 mRNA in bladder cancer samples as determined by RT-PCR. B, correlation between low miRNA-27a expression (red line) and anaggressive tumor phenotype as shown by progression to invasion and metastases following treatment in 139 freshly frozen tumors. C, high membranousSLC7A11 protein expression was identified in 10.5% of tumors [examples shown of high (100� and 630�) and absent expression (630�)] in a tissuearray of tumors from patients treated with adjuvant chemotherapy for invasive bladder cancer. D, SLC7A11 protein expression stratifies progression-freeand cancer-specific survival in patients treated with this agent within an RCT of cisplatin-based regimens. (�, P < 0.05; ��, P < 0.001).

Drayton et al.





interesting to see that our resistance mechanism was com-mon to all derived cells, regardless of initial population.Indeed, miRNA profiling revealed many other similaritiesbetween the resistant cell lines.Our findings have direct clinical implications for patient

care. First, analysis of tumor material revealed that miRNA-27a downregulation and associated SLC7A11 upregulationare not unique to a cell culture model of cisplatin resistanceand occur in tumors in vivo. Second, we identified thatcisplatin resistance could be reversed by either reinstate-ment of the miRNA, by siRNA-induced knockdown ofSLC7A11, or by inhibition of SLC7A11 with a small-mol-ecule inhibitor. Because the latter is possible with clinicallyachievable doses of sulfasalazine, our findings highlight theneed to evaluate this drug in combination with cisplatinwithin a clinical trial. Indeed, the side-effect profiles of theseagents suggest little cross-toxicity, beyond bone marrowsuppression. Third, the expression of miRNA-27a/27b andSLC7A11, or increased intracellular GSH within tumorsmay be used to predict cisplatin response in individualpatients. This could guide chemotherapy choice towardalternative agents not detoxified by GSH in tumors likelyto be resistant.Recent radiologic advances have enabled the spatial and

temporal resolution of glutamate (26) andGSH (35) in vivousing MRI spectroscopy. Our findings suggest that thisimaging modality could determine glutamate concentra-tions within a tumor and could be used as a surrogate forcisplatin resistance (36). This approach could inform theinitial chemotherapy regimen and monitor drug resistancebefore clinical relapse occurs.In summary, we have identified a novel mechanism of

cisplatin resistance in cancer namely increased expressionof mircoRNA-27a mediated regulation of intracellularGSH. The direct clinical implications of our work arethat this mechanism may be overcome by clinically

achievable doses of sulfasalazine and could be predictedusing MRI spectroscopy to measure tumor glutamateconcentrations.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: R.M. Drayton, E. Dudziec, H.E. Bryant, J.W.F.CattoDevelopment of methodology: R.M. Drayton, E. Dudziec, H.E. Bryant,J.W.F. CattoAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): R.M. Drayton, E. Dudziec, A. HartmannAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): R.M. Drayton, E. Dudziec, S. Peter,A. Hartmann, H.E. Bryant, J.W.F. CattoWriting, review, and/or revision of the manuscript: R.M. Drayton,E. Dudziec, A. Hartmann, H.E. Bryant, J.W.F. CattoAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): R.M. Drayton, S. Bertz, A. Hart-mann, J.W.F. CattoStudy supervision: J.W.F. Catto

AcknowledgmentsThe authors thank Emeritus Professor Shiro Bannai, University of Tsu-

kuba, Japan for assistance with A2780 cells, Dr. Gracjan Michlewski, Wel-come Trust Centre for Cell Biology for useful preliminarily investigations onmiRNA-27a processing, and ProfessorMarkMeuth for his helpful commentsin writing the manuscript.

Grant SupportJ.W.F. Catto was supported by a GSK Clinician Scientist fellowship and

project grants from Yorkshire Cancer Research (Grant number S305PA),Astellas Educational Foundation, and the European Union (European Com-munity’s Seventh Framework Programme, grant numbers: FP7/2007–2013,HEALTH-F2-2007-201438).H.E. Bryantwas supported by anRCUK researchfellowship.

The costs of publicationof this articleweredefrayed inpart by thepaymentofpage charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 11, 2013; revised November 25, 2013; accepted January20, 2014; published OnlineFirst February 10, 2014.

References1. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates

ofworldwide burden of cancer in 2008:GLOBOCAN2008. Int JCancer2010;127:2893–917.

2. Saxman SB, Propert KJ, Einhorn LH, Crawford ED, Tannock I, Ragha-van D, et al. Long-term follow-up of a phase III intergroup study ofcisplatin alone or in combination with methotrexate, vinblastine, anddoxorubicin in patients with metastatic urothelial carcinoma: a coop-erative group study. J Clin Oncol 1997;15:2564–9.

3. Pastore A, Federici G, Bertini E, Piemonte F. Analysis of glutathione:implication in redox and detoxification. Clin Chim Acta 2003;333:19–39.

4. Russo A, DeGraff W, Friedman N, Mitchell JB. Selective modulation ofglutathione levels in human normal versus tumor cells and subsequentdifferential response to chemotherapy drugs. Cancer Res 1986;46:2845–8.

5. Wu G, Fang YZ, Yang S, Lupton JR, Turner ND. Glutathione metab-olism and its implications for health. J Nutr 2004;134:489–92.

6. SatoH, TambaM, Ishii T, Bannai S.Cloning andexpressionof aplasmamembrane cystine/glutamate exchange transporter composed of twodistinct proteins. J Biol Chem 1999;274:11455–8.

7. Rose WC, Basler GA. In vivo model development of cisplatin-resistantand -sensitiveA2780humanovariancarcinomas. InVivo1990;4:391–6.

8. Drayton RM, Catto JW. Molecular mechanisms of cisplatin resistancein bladder cancer. Expert Rev Anticancer Ther 2012;12:271–81.

9. Drayton RM. The role of miRNA in the response to cisplatin treatment.Biochem Soc Trans 2012;40:821–5.

10. Olson P, Lu J, Zhang H, Shai A, Chun MG, Wang Y, et al. MiRNAdynamics in the stages of tumorigenesis correlate with hallmarkcapabilities of cancer. Genes Dev 2009;23:2152–65.

11. Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB. Pre-diction of mammalian miRNA targets. Cell 2003;115:787–98.

12. Pogribny IP, Filkowski JN, Tryndyak VP, Golubov A, Shpyleva SI,Kovalchuk O. Alterations of miRNAs and their targets are associatedwith acquired resistance ofMCF-7 breast cancer cells to cisplatin. Int JCancer 2010;127:1785–94.

13. Boren T, Xiong Y, HakamA,WenhamR, Apte S, ChanG, et al. MiRNAsand their target messenger RNAs associated with ovarian cancerresponse to chemotherapy. Gynecol Oncol 2009;113:249–55.

14. Liedert B, Pluim D, Schellens J, Thomale J. Adduct-specific mono-clonal antibodies for the measurement of cisplatin-induced DNAlesions in individual cell nuclei. Nucleic Acids Res 2006;34:e47.

15. Lehmann J, Retz M, Wiemers C, Beck J, Thuroff J, Weining C, et al.Adjuvant cisplatin plusmethotrexate versusmethotrexate, vinblastine,epirubicin, andcisplatin in locally advancedbladder cancer: results of a






randomized, multicenter, phase III trial (AUO-AB 05/95). J Clin Oncol2005;23:4963–74.

16. SmithSC,BarasAS,DancikG,RuY,DingKF,MoskalukCA, et al. A20-gene model for molecular nodal staging of bladder cancer: develop-ment and prospective assessment. Lancet Oncol 2011;12:137–43.

17. Nordentoft I, Dyrskjot L, Bodker JS,Wild PJ, HartmannA, Bertz S, et al.Increased expression of transcription factor TFAP2alpha correlateswith chemosensitivity in advanced bladder cancer. BMCCancer 2011;11:135.

18. Catto JW, Miah S, Owen HC, Bryant H, Dudziec E, Larre S, et al.Distinct miRNA alterations characterize high and low grade bladdercancer. Cancer Res 2009;69:8472–81.

19. Samimi G, Safaei R, Katano K, Holzer AK, Rochdi M, Tomioka M, et al.Increased expression of the copper efflux transporter ATP7Amediatesresistance to cisplatin, carboplatin, and oxaliplatin in ovarian cancercells. Clin Cancer Res 2004;10:4661–9.

20. Ishida S, Lee J, Thiele DJ, Herskowitz I. Uptake of the anticancer drugcisplatin mediated by the copper transporter Ctr1 in yeast and mam-mals. Proc Natl Acad Sci U S A 2002;99:14298–302.

21. Samimi G, Varki NM, Wilczynski S, Safaei R, Alberts DS, Howell SB.Increase in expression of the copper transporter ATP7A duringplatinum drug-based treatment is associated with poor survival inovarian cancer patients. Clin Cancer Res 2003;9:5853–9.

22. Hagrman D, Goodisman J, Souid AK. Kinetic study on the reactions ofplatinum drugs with glutathione. J Pharmacol Exp Ther 2004;308:658–66.

23. Masters JR, Thomas R, Hall AG, Hogarth L, Matheson EC, Cattan AR,et al. Sensitivity of testis tumour cells to chemotherapeutic drugs: roleof detoxifying pathways. Eur J Cancer 1996;32A:1248–53.

24. Buckingham SC, Campbell SL, Haas BR, Montana V, Robel S, Ogun-rinu T, et al. Glutamate release by primary brain tumors inducesepileptic activity. Nat Med 2011;17:1269–74.

25. Yuen TI, Morokoff AP, Bjorksten A, D'Abaco G, Paradiso L, Finch S,et al. Glutamate is associated with a higher risk of seizures in patientswith gliomas. Neurology 2012;79:883–9.

26. Cai K, Haris M, Singh A, Kogan F, Greenberg JH, Hariharan H, et al.Magnetic resonance imaging of glutamate. Nat Med 2012;18:302–6.

27. Zhuo L, Liu J,WangB,GaoM, HuangA. Differential miRNA expressionprofiles in hepatocellular carcinoma cells and drug-resistant sublines.Oncol Rep 2013;29:555–62.

28. Yang L, Li N, Wang H, Jia X, Wang X, Luo J. AlteredmiRNA expressionin cisplatin-resistant ovarian cancer cells and upregulation of miR-130a associated with MDR1/P-glycoprotein-mediated drug resis-tance. Oncol Rep 2012;28:592–600.

29. WangQ,ZhongM, LiuW, Li J,Huang J, ZhengL.AlterationsofmiRNAsin cisplatin-resistant human non-small cell lung cancer cells (A549/DDP). Exp Lung Res 2011;37:427–34.

30. Tang W, Zhu J, Su S, Wu W, Liu Q, Su F, et al. MiR-27 as a prognosticmarker for breast cancer progression and patient survival. PLoS ONE2012;7:e51702.

31. Fletcher CE, Dart DA, Sita-Lumsden A, Cheng H, Rennie PS, BevanCL. Androgen-regulated processing of the oncomir miR-27a, whichtargets Prohibitin in prostate cancer. Hum Mol Genet 2012;21:3112–27.

32. Scheibner KA, Teaboldt B, Hauer MC, Chen X, Cherukuri S, Guo Y,et al. MiR-27a functions as a tumor suppressor in acute leukemia byregulating 14–3-3theta. PLoS ONE 2012;7:e50895.

33. Nordentoft I, Birkenkamp-Demtroder K, Agerbaek M, Theodorescu D,Ostenfeld MS, Hartmann A, et al. miRNAs associated with chemo-sensitivity in cell lines and in advanced bladder cancer. BMC MedGenomics 2012;5:40.

34. Chen HH, Kuo MT. Role of glutathione in the regulation of Cisplatinresistance in cancer chemotherapy. Met Based Drugs 2010;2010.

35. SrinivasanR,RatineyH,Hammond-RosenbluthKE,Pelletier D,NelsonSJ. MR spectroscopic imaging of glutathione in the white and graymatter at 7 T with an application to multiple sclerosis. Magn ResonImaging 2010;28:163–70.

36. Diehn M, Cho RW, Lobo NA, Kalisky T, Dorie MJ, Kulp AN, et al.Association of reactive oxygen species levels and radioresistance incancer stem cells. Nature 2009;458:780–3.

Drayton et al.





2014;20:1990-2000. Published OnlineFirst February 10, 2014.Clin Cancer Res Ross M. Drayton, Ewa Dudziec, Stefan Peter, et al. SLC7A11in Bladder Cancer by Targeting the Cystine/Glutamate Exchanger Reduced Expression of miRNA-27a Modulates Cisplatin Resistance

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