A Possible Role for MicroRNA-141 Down-Regulation in Sunitinib

Resistant Metastatic Clear Cell Renal Cell Carcinoma Through

Induction of Epithelial-to-Mesenchymal Transition and

Hypoxia Resistance

Joost Berkers,* Olivier Govaere, Pascal Wolter,† Benoit Beuselinck, Patrick Schöffski,Léon C. van Kempen, Maarten Albersen, Joost Van den Oord, Tania Roskams,Johan Swinnen, Steven Joniau, Hendrik Van Poppel and Evelyne LerutFrom the Laboratory of Translational Cell and Tissue Research (JB, OG, JVdO, TR, EL) and Department of Oncology (JS), Catholic UniversityLeuven, Departments of Urology (JB, MA, SJ, HVP), Pathology (JVdO, TR, EL) and General Medical Oncology, Leuven Cancer InstituteLeuven (PW, BB, PS), University Hospitals Leuven, Leuven, Belgium, and Department of Pathology, McGill University/Jewish GeneralHospital (LCvK), Montreal, Quebec, Canada

Abbreviations

and Acronyms

ACTA1 � actin, � skeletalmuscle 1

AL � absolute luminescence

ATP � adenosine triphosphate

ccRCC � clear cell renal cellcarcinoma

CDH1 � E-cadherin

CDH2 � N-cadherin

EMT � epithelial-to-mesenchymaltransition

FN1 � fibronectin 1

KRT � keratin

miR � microRNA

NC � nontargeting control RNA

PCR � polymerase chain reaction

PFS � progression-free survival

PI � propidium iodide

qPCR � quantitative PCR

RT � real-time

TBST � tris buffered saline-Tween®

VIM � vimentin

ZEB � zinc finger E-box bindinghomeobox

Purpose: We identified microRNA driven mechanisms in clear cell renal cellcarcinoma associated with the tumor response to the multitargeted receptortyrosine kinase inhibitor sunitinib.Materials and Methods: We performed screening genome-wide microRNA real-time quantitative polymerase chain reaction on 20 freshly frozen clear cell renal cellcarcinoma tissue samples of patients who received sunitinib as first line targetedtherapy. Nine patients with progressive disease within 6 months after initiatingtherapy were considered poor responders and 11 with at least 1-year progression-free survival were considered good responders. We studied microRNA-141 functionin vitro by stable up-regulation of microRNA-141, quantification of target geneexpression and cell viability in normoxic and hypoxic conditions. Relative expressionin clinical and cell line samples was determined by real-time quantitative polymer-ase chain reaction. Localization of microRNA-141 and its targets was assessed bymicroRNA in situ hybridization and immunohistochemistry. Hypoxia induced cyto-toxicity was assessed by a luminescence adenosine triphosphate detection assay.Results: Compared to good responders, microRNA-141 was significantly down-reg-ulated in tumors of poor responders to sunitinib. This seemed spatially linked toepithelial-to-mesenchymal transition in vivo. Reintroduction of microRNA-141 in vitroreversed epithelial-to-mesenchymal transition and decreased cell viability in hypoxicconditions.Conclusions: In our study microRNA-141 down-regulation driven epithelial-to-mesenchymal transition in clear cell renal cell carcinoma was linked to anunfavorable response to sunitinib therapy. Reintroduction of microRNA-141 invitro led to epithelial-to-mesenchymal transition reversal and increased sensibil-ity to a hypoxic environment. Future experiments should be done in vivo to seewhether microRNA-141 driven reversal of epithelial-to-mesenchymal transitioncould affect the efficacy of sunitinib treatment.

Key Words: kidney; carcinoma, renal cell; MIRN141 microRNA, human;sunitinib; epithelial-mesenchymal transition

Accepted for publication November 26, 2012.Study received local ethical committee approval.* Correspondence: Minderbroederstraat 12, 3000 Leuven, Belgium (telephone: �32 16 336588; FAX: �32 16 336640; e-mail: johannes.

berkers@uzleuven.be).

† Financial interest and/or other relationship with Pfizer.

1930 www.jurology.com0022-5347/13/1895-1930/0 http://dx.doi.org/10.1016/j.juro.2012.11.133THE JOURNAL OF UROLOGY® Vol. 189, 1930-1938, May 2013© 2013 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH, INC. Printed in U.S.A.

MICRORNA-141 DOWN-REGULATION IN METASTATIC CLEAR CELL RENAL CELL CANCER 1931

IN Europe each year renal cell carcinoma develops in 3 to11/100,000 individuals with ccRCC being the most com-mon histological subtype.1 More than half of the patientswith ccRCC have metastasis during the disease course.2

In the metastatic setting, as we await the results ofCARMENA (Clinical Trial to Assess the Importance ofNephrectomy) comparing sunitinib vs nephrectomyfollowed by sunitinib, cytoreductive nephrectomy fol-lowed by systemic therapy is considered the standard ofcare based on the sequential benefit in the immunother-apy era.3,4 Sunitinib, a multitarget receptor tyrosine ki-nase inhibitor that mainly targets vascular endothelialgrowth factor receptor, reduces the tumor burdenthrough hypoxia induced tumor cell death. In thesunitinib registration trial half of the treated pa-tients with a favorable or intermediate risk scorebased on Memorial Sloan-Kettering Cancer Centercriteria achieved an objective response, resulting ina median PFS of 11 months.5,6 Nevertheless, in arelevant proportion of treated patients the clinicalbenefit in PFS remains limited. Moreover, eventu-ally all patients experience relapse due to acquiredresistance or other reasons. Identifying pathwaysresponsible for intrinsic or acquired resistance couldprovide novel directions to develop therapies thatblock resistance pathways. Although different resis-tance mechanisms have been proposed, reliable bio-markers predictive of sunitinib sensitivity or prima-ry/secondary resistance are still lacking.7

Studies are needed to clarify the mechanisms thatmake ccRCC unsusceptible or susceptible to sunitinibtherapy and identify predictive biomarkers of asunitinib response in a given patient population.8

miRs regulate gene expression by inhibiting the trans-lation and promoting the degradation of complementarymRNA.9 They modulate crucial biological processes, suchas differentiation, proliferation and apoptosis. Deregu-lated miRs have been reported in all urological malig-nancies.10 miRs interact with the hypoxia-inducible fac-tor signaling pathway, which is the main deregulatedpathway in ccRCC due to inactivation of the von Hippel-Lindau gene.11 miR based therapeutic applications areemerging that may be used to augment the response toreceptor tyrosine kinase inhibitor-I in the future.12

We linked miR expression with the clinical bene-fit of sunitinib treatment and the molecular charac-teristics of metastasized ccRCC.

MATERIALS AND METHODS

Patients with Metastatic ccRCCFrom a single institutional database (2006 to 2010 atUniversity Hospitals Leuven) we selected cytoreductivenephrectomy specimens from targeted treatment naïvepatients 1) who received sunitinib treatment as first linetargeted therapy, and 2) in whom review of paraffin em-

bedded and frozen tissue by a single experienced uro-

pathologist (EL) confirmed ccRCC (greater than 90% via-ble tumor tissue), grade and stage. Only patients withsynchronous metastasis were selected. Patients received50 mg sunitinib daily according to the manufacturer 4weeks on/2 weeks off schedule. To detect clinically mean-ingful differences, 2 clinical outcome extremes were usedas selection criteria for initial genome-wide miR RT-qPCRanalysis. The 11 patients with PFS greater than 1 yearwere considered good responders and the 9 with progres-sive disease within 6 months were considered poor re-sponders. Tumor evaluation was based on RECIST1.1.13

Local ethical committee approval was obtained for the useof clinical data and the remaining tissue from nephrec-tomy specimens for study purposes.

Cell CulturesThe UMRC-2 and RCC4 cell lines (European Collection ofCell Cultures, Salisbury, United Kingdom), derived fromprimary human ccRCC, were selected for in vitro experi-ments. They were cultured in Advanced Dulbecco’s Modi-fied Eagle’s Medium (Gibco®) with 10% heat inactivatedfetal bovine serum and L-glutamine (Invitrogen™) at 37Cin a humidor supplemented with 5% CO2.

Genome-Wide miR RT-qPCR

in Human Tissue SamplesmiRs were isolated from 20 fresh frozen ccRCC samplesusing the RT2 qPCR-Grade miRNA Isolation Kit (SA Bio-sciences™). Reverse transcription was done using the RT2

miRNA First Strand Kit (SA Biosciences) according to thesupplier protocol. We quantified 752 known miRs withgenome-wide 2 � 384-well based RT2 miRNA PCR Arrays(SA Biosciences) on an ABI 7900HT System (Applied Bio-systems™) using RT2 SYBR® Green/ROX PCR MasterMix (SA Biosciences). Cycle threshold data were nor-malized to reference small RNA (U6, and SNORD44, 47and 48). The fold change, calculated using the equation,2ˆ(���Ct), where Ct represents the cycle threshold, was

Table 1. Clinical and pathological characteristics of 20 patientswith metastasized stage IV ccRCC selected for whole genomemiR qPCR analysis

Poor Response Good Response p Value

No. pts 9 11Median days survival (95% CI):

PFS 81 (33–129) 728 (505–951) �0.0001Overall 185 (96–275) 1,062 (762–1,363) �0.0001

Mean age at diagnosis (95% CI) 58 (52–65) 57 (52–62) 0.94No. interferon beforesunitinib (%)

4 (44) 6 (55) 1

Median No. Memorial Sloan-Kettering Cancer Center riskfactors (IQR)

2 (1–3) 2 (0–3) 0.22

Mean cm primary tumordiameter (95% CI)

113 (87–138) 84 (49–119) 0.17

Median Furhman grade (IQR) 4 (3–4) 3 (3–4) 0.11No. T stage (%):

2 or Less 0 2 (18)3 9 (100) 8 (73) 0.604 0 1 (9)

No. sarcomatoid features (%) 1 (11) 2 (18) 0.77No. regional lymph node 3 (33) 2 (18) 0.43

involvement (%)

MICRORNA-141 DOWN-REGULATION IN METASTATIC CLEAR CELL RENAL CELL CANCER1932

determined using RT2 Profiler Online Data Analysis (SABiosciences).

Cell Culture Sample RT-qPCRCell line samples were lysed with RLT buffer (Qiagen®)containing 1% (volume per volume) �-mercaptoethanol(Sigma-Aldrich®). mRNA was purified using the RNeasy®Mini Kit. Reverse transcription was performed with theiScript™ cDNA Synthesis Kit. RT-PCR arrays were per-formed on an ABI 7900HT System using Fast SYBR GreenMaster Mix (Applied Biosystems®). Primers were custommade using PerlPrimer (http://perlprimer.sourceforge.net/)or obtained from RTPrimerDB (http://medgen.ugent.be/rtprimerdb/). We determined the expression of the FN1,ACTA1, VIM, CDH2, CDH1, KRT7, KRT19, ZEB1 andZEB2 genes. Cycle threshold data were normalized to thereference genes glyceraldehyde-3-phosphate dehydrogenase,hypoxanthine phosphoribosyltransferase 1 and ribosomalprotein L19.

Figure 1. A to F, spatial correlation of miR-141 and EMT markeexpression. D, high nuclear and cytoplasmic expression. B anmembranous positivity. C and F, immunohistochemical staining

Table 2. Differentially expressed miR and corresponding functitherapy compared to that in 11 with favorable control

Fold p Value

Up-regulated:miR-520g 7.25 0.036 Inhibits nuclearmiR-155 6.72 0.040 Promotes invasmiR-526b 4.80 0.0067 Unknown

Down-regulated:miR-144 �4.77 0.016 Inhibits NRF2 wmiR-141 �4.03 0.0098 Inhibits ZEB tra

mitogen activainducing chem

miR-376b �4.00 0.032 Unknown

cells. Reduced from �200.

In Situ HybridizationParaffin sections from the 20 nephrectomy specimenswere digested with 5 �g/ml proteinase K (Sigma-Aldrich)in 20 mM tris/5 mM ethylenediaminetetraacetic acid(Dako®) (pH 7.4) for 15 minutes at 37C. After washingwith phosphate buffered saline (Dako), tissue was cross-linked in 4% formaldehyde/phosphate buffered saline for 5minutes. Nonspecific sites were blocked by incubation in0.25% acetic anhydride in 0.1M triethanolamine (1:1) (In-vitrogen) for 2 � 5 minutes at room temperature. Slideswere washed with saline sodium citrate (Invitrogen) for2 � 5 minutes and air dried at 37C. They were incu-bated overnight with miR-141 3= end digoxigenin la-beled locked nucleotide acid detection probes (Exiqon,Vedbæk, Denmark) diluted in hybridization buffer com-posed of 4 � saline sodium citrate, 1 � Denhardt solu-tion, 1 ng/ml tRNA, 0.01M dithiothreitol, 0.25 mg/mlherring sperm DNA (Sigma-Aldrich) and 10% dextransulfate (Invitrogen) at a probe specific melting temper-

ession in ccRCC. A and D, miR-141 in situ hybridization. A, lowmunohistochemical staining for CDH1. B, negative. E, strong

M. C, strong cytoplasmic positivity. F, negative results in tumor

atients with poor disease control on sunitinib targeted

Function

B signaling and inflammatory response in breast Ca14

epatocellular Ca � inhibits apoptosis in leukemia15

sequent decrease in oxidative stress tolerance in sickle cell disease16

n factors, promoting epithelial phenotype in solid tumors,17,18 � inhibits p38�tein kinase with subsequent increased sensitivity to reactive oxygen speciesy in ovarian Ca19

r exprd E, imfor VI

on in p

factor-�ion in h

ith subnscriptioted prootherap

nifican

MICRORNA-141 DOWN-REGULATION IN METASTATIC CLEAR CELL RENAL CELL CANCER 1933

ature of �20C in a humidified chamber. U6 targetingand scrambled probes served as positive and negativecontrols, respectively.

Slides were washed and pre-incubated with 2% normalsheep serum in TBST for 15 minutes at room temperatureand incubated with sheep anti-DIG-alkaline phosphatase(1:1,000 in 2% normal sheep serum/TBST) (Roche, Basel,Switzerland) for 60 minutes at room temperature. Afterwashing with TBST and 0.1M tris-HCl 3 � 5 minutes (pH9.5) and incubation at 37C in BM Purple (Roche), theslides were counterstained with nuclear fast red.

Expression was independently evaluated by 2 experi-enced researchers. In case of disagreement consensus wasachieved by discussion. Localization was determined asnuclear, cytoplasmic or membranous. Semiquantitativeevaluation of intensity (negative, weak-moderate orstrong) was assigned compared with internal controls innormal renal cortex (distal convoluted tubules).

ImmunohistochemistryPatient samples. Immunohistochemistry was performedfor CDH1 (1:50) and VIM (1:500) (Dako), each for 30minutes at room temperature, on serial paraffin sections

Figure 2. A, cells transduced with miR-141 containing vector stransduced with vector containing NC and untreated cells. AsterqPCR was apparent in miR-141 transduced cells vs NC. Doubsignificant decrease was found in ZEB1 expression. NS, not sig

of the 20 nephrectomy specimens. Antigen retrieval was

done using EnVision™ FLEX Target Retrieval Solution,endogenous peroxidase activity was blocked using Perox-idase Blocking Reagent (Dako) and visualization was donewith the EnVision™ Dual Link Kit and 3,3=-diaminoben-zidine (Dako). Quantification was performed as describedfor in situ hybridization. Internal controls were distalconvoluted tubules for CDH1 and endothelial cells forVIM.

Cell cultures. Immunohistochemistry for CDH1 and VIMwas performed on cultured and transduced UMRC-2 cellsseeded in Millicell® EZ Slides. After 96 hours, the cellswere fixed in acetone for 8 minutes and incubated withprimary antibody for 30 minutes at room temperature.Visualization was achieved using UltraVision LP Detec-tion System HRP Polymers (ThermoFisher Scientific, Fre-mont, California) and 3-amino-9-ethylcarbazole (Dako).

Stable miR-141 Over ExpressionUMRC-2 and RCC4 cells were transduced with a lentivi-ral vector containing miR-141 or nonsilencing controlRNA linked to a cytomegalovirus promoter and 2 con-structs realizing puromycin resistance and TurboGFP

significantly increased miR-141 expression on qPCR vs cellsicates p � 0.0001. B, significant decrease in ZEB2 expression onrisks indicate p �0.01. Triple asterisks indicate p � 0.01. Not (UMRC p � 0.23 and RCC4 p � 0.06).

howedisk indle aste

(Thermo Scientific, Huntsville, Alabama) expression. At

MICRORNA-141 DOWN-REGULATION IN METASTATIC CLEAR CELL RENAL CELL CANCER1934

48 hours after transduction at a multiplicity of infection of1 the culture medium was enriched with puromycin(5 �g/ml) to select stable integrants. Successfully trans-duced cells were selected by fluorescence activated cellsorting based on TurboGFP expression. As controls, weused cells transduced with a vector containing NC as wellas untreated cells. miR-141 RT-qPCR was performed witha miScript miR-141 primer assay (Qiagen), as described.

Cell Viability AssayAt 24 hours after seeding UMRC-2 and RCC4 cells, trans-duced (miR-141 or NC) and untreated cells were placed inhypoxic (1% O2) or normoxic conditions for 48 hours. Cellswere lysed and the ATP present was quantified by theluciferase based ATPlite 1step® Luminescence Assay Sys-tem. AL was read with a Luminoskan Ascent MicroplateReader (Thermo Scientific). Relative luminescence wasdefined as AL in hypoxic conditions/AL in normoxic con-ditions.

Flow Cytometric Quantification of Cell DeathAt 24 hours after seeding, miR-141 and negative controltransduced UMRC-2 and RCC4 cells were placed in hy-poxic (1% O2) or normoxic conditions for 12, 24, 48 and 96hours, respectively. Cells were resuspended in the originalculture medium to include nonadherent cells and centri-fuged. Pellets were suspended in binding buffer (BDPharmingen™) at 1 � 106 cells per ml. Solution (100 �l)was incubated with PI for 15 minutes and 400 �l bindingbuffer were added before flow cytometry. PI positive cellswere counted and described as percent of the total cellcount per sample. Measurements were made in triplicate.

Statistical AnalysisThe nonparametric Mann-Whitney U, parametric Stu-dent t or chi-square test, or ANOVA was used for sta-tistical analysis depending on data distribution withGraphPad® software. Statistical significance was con-sidered at p �0.05.

RESULTS

Genome-Wide miR RT-qPCR

on Human ccRCC Samples

Genome-wide miR RT-qPCR was performed on 20samples, including 9 from poor responders and 11from good responders. Except for the significantlydifferent PFS/overall survival after more than 18months of followup, the clinical and pathologicalcharacteristics of the 2 groups were well balanced(table 1). Table 2 lists statistically significantly dif-ferent expressed miRs with a threshold cycle of 35 orless, and greater than threefold regulation changesand function.14–19 Significant miR-141 down-regula-tion was noted in poor responders (p � 0.0098).

Of the features described only sarcomatoid fea-tures in the tumor correlated with significantlylower miR-141 expression (mean � SD fold expres-sion 4.8 � 4.8 vs 57.2 � 77, p � 0.034, table 1).Interferon treatment was not associated with differ-

ently expressed miR-141 (data not shown).

miR-141

Intracellular localization and relation to targeted

pathways in human tissue samples. In situ hybrid-ization revealed that miR-141 was variably ex-pressed in different regions in 1 ccRCC, most fre-quently more intense in the peripheral portion of thetumor. Cytoplasmic and nuclear expression wasnoted. No correlation was found with Fuhrmangrade. In accord with qPCR data, low expressionwas noted in sarcomatoid dedifferentiated tumorareas in 3 cases.

Tumor regional positivity for miR-141 spatiallycorrelated positively with immunohistochemicalCDH1 expression and negatively with VIM expres-sion (fig. 1). Due to high spatial and interpatientintensity variability of the miR in situ hybridizationsignal, patients could not be reliably categorizedquantitatively.

Except in endothelium, inflammatory and stro-mal cells were miR-141 negative. In the surroundingnontumor renal cortex miR-141 positivity was most

Figure 3. EMT reversal in vitro via miR-141 over expression. InmiR-141 transduced UMRC-2 and RCC4 cells mRNA levels ofmesenchymal markers ACTA1, VIM, CDH2 and FN1 were de-creased, and mRNA levels of epithelial markers CDH1, and KRT7and 19 were increased. Bar graph shows log2-fold regulationratios of miR-141 transduced/NC transduced cells. Red barsrepresent mesenchymal cells. Green bars represent epithelialcells.

prominent in the distal tubules.

d �10

MICRORNA-141 DOWN-REGULATION IN METASTATIC CLEAR CELL RENAL CELL CANCER 1935

In vitro over expression and effect on its targeted

pathways. Stable transduction of UMRC-2 andRCC4 cells with a miR-141 containing vector led toincreased miR-141 expression on qPCR comparedto that of negative control samples (p � 0.0001,fig. 2, A). Subsequent qPCR analysis for ZEB tran-scription factors confirmed a miR-141 induced de-crease in ZEB expression, particularly ZEB2 (p �0.01,fig. 2, B).

miR-141 induced inhibition of ZEB transcriptionfactors resulted in reversed EMT markers on themRNA level, including decreased expression of themesenchymal markers FN1, ACTA1, VIM andCDH2, and increased expression of the epithelialmarkers KRT19, CDH1 and KRT7 in miR-141 trans-duced UMRC2 cells compared to negative controls(fig. 3).

miR-141 instigated EMT reversal was confirmedon the protein level. Immunohistochemical expres-sion of VIM and CDH1 was decreased and increased,respectively, in miR-141 transduced cells (fig. 4).

Cell Viability in Normoxic

and Hypoxic Conditions

miR-141 over expression in the UMRC-2 and RCC4cell lines resulted in significantly decreased cell vi-ability in normoxic conditions. Moreover, after cor-recting for the decreased proliferation rate in nor-moxic conditions, we noted increased susceptibilityto hypoxia in miR-141 transduced cells (fig. 5, A andB). To rule out increased cell death as the cause ofdecreased cell viability in miR-141 transduced cells,

Figure 4. Changes in expression of epithelial and mesenchymachemical staining for CDH1. A, high expression in miR-141 transcells. D to F, immunohistochemical staining for VIM. D, low extransduced and untreated cells. Reduced from �200 (A to C) an

cell death was quantified after exposure to hypoxia

using percent of PI positive cells as a surrogate forcell death. An increase in PI positive cells was ob-served after exposure to hypoxia but there was nodifference in miR-141 and NC (fig. 5, C and D). Thissuggested that the decreased viability of miR-141transduced cells in hypoxic circumstances maymainly originate from hypoxia induced decreasedproliferative capacity (fig. 5).

DISCUSSION

We explored whether quantitative miR expressioncorrelated with the clinical and molecular behaviorof metastasized ccRCC in response to sunitinib.Therefore, we studied miR expression in cytoreduc-tive nephrectomy specimens of metastatic ccRCCtumors using a quantitative miRNA PCR primerarray platform. Two study populations were definedbased on PFS on sunitinib. Tumors with an unfavor-able response (PFS less than 6 months in 9 patients)were characterized by a number of significantly de-regulated miRs compared to tumors with favorabledisease control (PFS greater than 1 year in 11 pa-tients).

Interestingly, one of the differentially ex-pressed miRs was miR-141, which was signifi-cantly down-regulated in the patient populationwith unfavorable PFS. Also, in this small studypopulation sarcomatoid features were associatedwith miR-141 down-regulation. We focused onmiR-141 since multiple groups have reported itsrole as a part of the miR-200 family with a pivotal

ers induced by miR-141 in UMRC-2 cells. A to C, immunohisto-cells. B and C, low expression in NC transduced and untreatedn in miR-141 transduced cells. E and F, high expression in NC0 (D to F).

l markduced

pressio

role in tumor progression.17–19 The miR-200 family

MICRORNA-141 DOWN-REGULATION IN METASTATIC CLEAR CELL RENAL CELL CANCER1936

represses EMT in several tumor types20,21 and innoncancerous renal epithelium.22,23 miR-141 inhib-its activity of the transcriptional repressors ZEB1and 2, which are factors previously implicated inEMT, tumor metastasis, angiogenesis induction andchemotherapy resistance.24 We confirmed the in-volvement of miR-141 in EMT since reintroducingmiR-141 in the ccRCC cell lines was associated withZEB2 down-regulation. We found the same result inour human sample set, in which ZEB2 expression in

Figure 5. A, significantly decreased cell viability was seen in m(NC). Cell viability was defined by ATP quantification using lucifeasterisks indicate p �0.01. B, significant hypoxia induced decreavs negative control and untreated cells. Cell viability in hypoxicassay after 48 hours of 1% O2 hypoxia. Relative luminescence (Aof miR-141 transduced cells in normoxic condition. Single asteriDouble asterisks indicate p � 0.026 vs normal control and p �end stage apoptotic and death cells based on PI positivity. C, sigconditions at 48-hour time point analysis. Single asterisk indicatep � 0.05. Quadruple asterisks indicate p � 0.02. D, no significanconditions at different time point analyses. h, hours.

renal cell carcinoma samples from poor responders

was higher than in samples from good responders,although not significantly so (p � 0.07, data notshown), probably due to the substantial SD of thesmall sample size. Similarly, the subsequent rever-sal of EMT on mRNA (qPCR) and protein level (im-munohistochemistry) in vivo was not as strong as itwas in vitro.

An additional explanation could be due to theclear cell morphology of the tumor cells. ccRCC cellstypically accumulate lipid and glycogen, which dis-

transduced UMRC-2 and RCC4 cells (141) vs negative controlased assay at 48 hours. Single asterisk indicates p �0.01. Doubleability was seen in miR-141 transduced UMRC-2 and RCC4 cellsnment was defined by ATP quantification with luciferase baseda/ALnormoxia) is reported to correct for already decreased viabilityicates p � 0.025 vs normal control and p � 0.0037 vs untreated.vs untreated. C, and D, flow cytometric quantitative analysis oft increase in percent of PI positive cells in hypoxic vs normoxic

0.02. Double asterisks indicate p � 0.05. Triple asterisks indicaterence in PI positivity by miR-141 status in normoxic or hypoxic

iR-141rase bsed vienviroLhypoxi

sk ind0.017nificans p �t diffe

solve during tissue processing, resulting in an opti-

MICRORNA-141 DOWN-REGULATION IN METASTATIC CLEAR CELL RENAL CELL CANCER 1937

cally clear or empty cytoplasm on hematoxylin andeosin staining. This empty cytoplasm might be re-sponsible for weak cytoplasmic staining and difficultinterpretation on in situ hybridization and immuno-histochemistry. Although decreased miR-141 ex-pression was reported in ccRCC compared to that inadjacent nontumor renal cortex,20–22 to our knowl-edge this is the first report of a correlation betweenmiR-141 and ccRCC clinical behavior in response tosunitinib treatment. However, since our data arebased on a small homogeneous population of pa-tients with synchronous metastases who underwentcytoreductive nephrectomy, the correlation cannotyet be extrapolated to the general population of pa-tients with metastatic ccRCC. Furthermore, in situhybridization revealed variable degrees of miR-141expression at different regions of 1 tumor sample.This is consistent with the extensive degree of ge-netic intratumor heterogeneity present in ccRCC.25

Therefore, larger study populations are needed toexclude sampling bias as a possible explanation forthe differential miR expression between the studygroups.

To our knowledge how miR-141 down-regulationis mediated remains to be elucidated. Since thegenomic location on chromosome 12 (12p13.31) isrich in CpG islands, it is potentially affected byhypermethylation, consequently inhibiting expres-sion.26

Unlike classic chemotherapy, the antitumor effectof sunitinib for ccRCC is not achieved by cytotoxicitybut by promoting apoptosis since it induces a hy-poxic environment due to angiogenesis inhibi-tion.27–29 Tumors in our poorly responding patients

showed the characteristics of EMT, as possibly ex-

REFERENCES

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We observed augmented sensitivity to a hypoxicenvironment in vitro after introducing miR-141 ex-pression in ccRCC cell lines, inducing decreased pro-liferative capacity. This suggests that miR-141down-regulation could have a guiding role in EMTrelated tumor hypoxia resistance. These data sup-port the notion that miR-141 might be important forccRCC proliferative pathways and an important tar-get in the battle against drug resistance. However,the exact mechanism of hypoxia resistance in ccRCCthat expresses low miR-141 remains to be eluci-dated.

CONCLUSIONS

In this study miR-141 down-regulation driven EMTin ccRCC was linked to an unfavorable response tosunitinib. miR-141 reintroduction in vitro led toEMT reversal and increased sensitivity to hypoxia.Future in vivo experiments should be done to deter-mine whether miR-141 driven reversal of EMT af-

fects sunitinib efficacy.

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