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Cancer Biology and Signal Transduction

c-Jun N-Terminal Kinase Inactivation by Mitogen-Activated Protein Kinase Phosphatase 1Determines Resistance to Taxanes andAnthracyclines in Breast CancerRa�ul Rinc�on1, Sandra Zazo1, Cristina Chamizo1, Rebeca Manso1, Paula Gonz�alez-Alonso1,Ester Martín-Aparicio1, Ion Crist�obal2, Carmen Ca~nadas1, Rosario Perona3, Ana Lluch4,Pilar Eroles4, Jes�us García-Foncillas2, Joan Albanell5,6,7, Ana Rovira5,6,Juan Madoz-G�urpide1, and Federico Rojo1

Abstract

MAPK phosphatase-1 (MKP-1) is overexpressed duringmalignant transformation of the breast in many patients, andit is usually associated with chemoresistance through interfer-ence with JNK-driven apoptotic pathways. Although the molec-ular settings of the mechanism have been documented, detailsabout the contribution of MKP-1 to the failure of chemother-apeutic interventions are unclear. Transient overexpression ofMKP-1 and treatment with JNK-modulating agents in breastcarcinoma cells confirmed the mediation of MKP-1 in theresistance to taxanes and anthracyclines in breast cancer,through the inactivation of JNK1/2. We next assessed MKP-1expression and JNK1/2 phosphorylation status in a large cohortof samples from 350 early breast cancer patients treated with

adjuvant anthracycline–based chemotherapy. We detected thatMKP-1 overexpression is a recurrent event predominantlylinked to dephosphorylation of JNK1/2 with an adverseimpact on relapse of the tumor and overall and disease-freesurvival. Moreover, MKP-1 and p-JNK1/2 determinationsin 64 locally advanced breast cancer patients treated withneoadjuvant taxane–based chemotherapy showed an inversecorrelation between MKP-1 overexpression (together withJNK1/2 inhibition) and the pathologic response of the tumors.Our results emphasize the importance of MKP-1 as a potentialpredictive biomarker for a subset of breast cancer patientswith worse outcome and less susceptibility to treatment. MolCancer Ther; 15(11); 2780–90. �2016 AACR.

IntroductionImproving breast cancer therapy requires novel prognostic and

predictive markers (1, 2). The prognostic labeling of this hetero-geneous disease is still based on conventional tumor–node–metastasis (TNM) staging and histopathologic features (3, 4).While estrogen receptor (ER)–dependent and progesterone recep-

tor (PR)–dependent tumors and HER2-positive subtypes havebeen approved for noncytotoxic regimens, the triple-negativebreast cancer subtype lacks a therapy alternative to chemotherapy(5). Hence, the molecular dynamics that rule breast cancer path-ogenesis need to be elucidated to develop precise therapiesto enhance survival and overcome resistance to standard chemo-therapy regimens (6, 7).

MAPKs have been extensively described as some of the keymolecular events driving breast cancer progression (8, 9). Part ofcancer-related MAPK regulation consists of a dual dephosphor-ylation performed by MAPK phosphatases (MKP; ref. 8). Amongthem, MKP-1 plays a relevant role in tumorigenesis, being able todephosphorylate all MAPKs, with substrate preference for p38MAPK and c-Jun N-terminal kinase (JNK). The activation of theJNK pathway has been linked to the apoptosis induced by severalchemotherapeutic agents. In breast cancer, JNK dephosphoryla-tion has been correlated with cancer progression and tumorsurvival against different stress conditions, such as chemotherapyor oxidative damage (10–13). Further experimental research hasrelated MKP-1 with tumor response to stress in breast cancer. Ofimportance, a tuned MAPKs/MKPs balance regulates cellularresponse to cancer therapy, as revealed by experimental evidence.For instance, in breast cancer cells, doxorubicin is able to activateJNK pathway to achieve its antitumoral effect (14, 15). On thecontrary,MKP-1mediates different tumor responses to anticancertherapy, depending on its activity or inactivity: MKP-1 activates

1Pathology Department, IIS-Fundaci�on Jim�enez Díaz, UAM, Madrid,Spain. 2Translational Oncology Division, Oncohealth Institute, HealthResearch Institute FJD-UAM, University Hospital Fundaci�on Jim�enezDíaz, Madrid, Spain. 3"Alberto Sols" Biomedical Research InstituteCSIC-UAM, Madrid, Spain. 4Institute of Health Research INCLIVA,Valencia, Spain. 5Medical Oncology Department, Hospital del Mar,Barcelona, Spain. 6Cancer Research Program, IMIM (Hospital del MarResearch Institute), Barcelona, Spain. 7Universitat Pompeu Fabra,Barcelona, Spain.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Authors: Juan Madoz-G�urpide, IIS-Fundaci�on Jim�enez Díaz,UAM, Avda. Reyes Cat�olicos 2, E-28040 Madrid, Spain. Phone: 34-915504800; Fax: 34-915448246; E-mail: [email protected]; or Federico Rojo,Pathology Department, University Hospital Fundaci�on Jim�enez Díaz, Avda.Reyes Cat�olicos 2, E-28040 Madrid, Spain. Phone: 34-915504800; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-15-0920

�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

Mol Cancer Ther; 15(11) November 20162780

on September 11, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

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antiapoptotic pathways in response to proteasome inhibitors(16, 17) as well as antiproliferative activity to PR in breast cancercells (18). Of relevance, MKP-1 inhibition by small moleculesenhanced the antitumoral effect of paclitaxel (19), and transientexpression modulation of MKP-1 defined breast cancer cells'ability to survive after exposure to different cytotoxic agents(13). Given the reported apoptotic role of JNK in response totaxanes- and anthracyclines-based therapies, as well as the abilityof MKP-1 to decrease JNK activation, we decided to explore theinterplay of this kinase/phosphatase pair in the resistance ofbreast cancer cells to docetaxel and doxorubicin.

Previously, we showed that MKP-1 was overexpressed duringthe malignant transformation of the breast, thus affecting MAPKexpression, and its activation could be inhibited by doxorubicintreatment (15). Nevertheless, we discovered that breast tumorsoverexpressing MKP-1 did not show this MAPK alteration afterthis treatment (15). In the present study, we confirm the impor-tance of MKP-1 overexpression as a negative prognostic marker ofresponse to chemotherapy. Of importance, we demonstrate thatdocetaxel and doxorubicin regulate ERK1/2 and JNK1/2 activa-tion in part through MKP-1 modulation. Further, MKP-1 over-expression dephosphorylates JNK1/2 and results in higher cellgrowth and lower apoptotic rates in the tumor cells.

Materials and MethodsCell cultures and reagents

MDA-MB-231 (ATCC HTB-26) and BT-474 (ATCC HTB-20)cell lines were purchased from the American Type Culture Col-lection (ATCC) and authenticated according to the requiredstandards (LGC Standards). Docetaxel, doxorubicin, anisomycin,and SP6000125 were purchased from Sigma Aldrich. HumanMKP-1 cDNA was purchased from Open Biosystems and clonedinto a pBluescriptR vector (clone ID 4794895; Open Biosystems,Dharmacon), digested with EcoRI and KpnI enzymes, andinserted into a pCMV-HA plasmid. Transfections were carriedout using Lipofectamine 2000 (Life Technologies) and followingthe manufacturer's indications.

Cell assaysCell proliferation was measured in triplicate by MTS cell

proliferation assay using the CellTiter 96 AQueous One SolutionCell Proliferation Assay (Promega), following the manufacturer'sindications. Cell growthwas analyzed in triplicate by crystal violetassay as previously reported (20). Apoptosis was measured usingAnnexin V FITC Apoptosis Detection Kit I (BD Biosciences) andquantified in a FACS CANTO II cytometer (BD Biosciences).

Western blotting analysisWestern blotting (WB) analysis in protein extracts from cul-

tured cells was done as previously reported (21). Antibodies wereas follows: anti–phospho-ERK1/2 (p-ERK1/2; Thr202/Tyr204),anti-ERK1/2, and anti-JNK (Cell Signaling Technology); anti-active JNK pAb (p-JNK; Thr183/Tyr185; Promega); anti-MKP-1(Santa Cruz Biotechnology); anti–a-tubulin and anti-GAPDH(Sigma-Aldrich); and anti-Rabbit IgG (GE Healthcare).

Microarray analysisTotal RNA from the cell lines was isolated using the RNeasy

mini Kit (Qiagen). RNApurity and integrity were assessed both byspectrophotometry (NanoDrop ND-2000, NanoDrop Technolo-gies) and electrophoresis (2100 Bioanalyzer, Agilent Technolo-

gies); for microarray experiments, the minimal requirements forRNApuritywereA260/280>2.0 andA260/230>1.4 andRIN>9.4.Microarray expression profiles were obtained using the AffymetrixGeneChip Human Exon 1.0 ST Array (Affymetrix Inc.). Followinghybridization, the array was stained in the Affymetrix GeneChipFluidics Station 450 and scanned using a GeneChip Scanner3000 7G.

Gene expression profile analysisData were processed following the methodology previously

described (22). Briefly, after quality control of raw data, back-ground was corrected, quantile-normalized, and summarized tothe gene level using the robust multi-chip average. Only thosetranscripts with an intensity signal of more than 10% of allintensities of the mean of studied groups and then over 50% ofvariance from total resting variance were considered for furtheranalysis. Linear Models for Microarray (LIMMA) were used fordetecting differentially expressed genes between conditions. Cor-rection for multiple comparisons was performed using the FDR,and only genes with an adjusted P value <0.05 were selected assignificant. For the purposes of functional analysis, genes wereselected to have an unadjusted P value <0.05. Hierarchical clusteranalysis was also performed. All data analysis was performed in Rwith the packages aroma.affymetrix, Biobase, LIMMA, and gene-filter. Functional analysis was performed with Ingenuity PathwayAnalysis software (Ingenuity Systems). All microarray procedureswere performed by the IMIM Microarray Core Facility (SAM).

Quantitative real-time PCRcDNA was produced using the Universal Transcriptor cDNA

synthesis Kit (RocheDiagnostics) according to themanufacturer'srecommendations. MKP-1 gene expression levels were deter-mined using a quantitative real-time PCR (qPCR) assay, withATP5E as a housekeeping gene. Primers were designed using theLasergene Primer design software (DNASTAR Inc.) and based onthe following genomic sequences: MKP-1 (NM_004417.3) andATP5E (NM_006886.3). qPCRs were performed using the Light-Cycler480 II system (RocheApplied Science) for 45 cycleswith thefollowing set of primers:MKP-1, Fw, 50-GAGGCCATTGACTTCA-TAGAC-30 and Rv 50-GTAAGCAAGGCAGATGGTG-30;ATP5E, Fw,50-GTAGCTGAGTCCAGCCTGTC-30 and Rv 50- GATCTGGGAG-TATCGGATG-30. Specific probes from theUniversal Probe Library(Roche Applied Science) were selected. Relative gene expressionlevels (RQ) were calculated in accordance with the MIQE guide-lines (23).

Patient samplesThree-hundred and fifty surgically resected specimens from

primary breast tumors were obtained from Parc de Salut MarBiobank (MARBiobanc), Fundaci�on Jim�enez Díaz Biobank, andValencia Clinic Hospital Biobank. Tumor specimens from for-malin-fixed paraffin-embedded (FFPE) blocks were retrospective-ly selected from consecutive breast cancer patients diagnosedbetween 1998 and 2000, following these criteria: infiltratingcarcinomas, operable, no neoadjuvant therapy, sufficient avail-able tissue, and clinical follow-up. TNM staging was classifiedusing the American Joint Committee on Cancer staging system.Histologic grade was defined according to the Elston-Ellis mod-ification of the Scarff–Bloom–Richardson grading system (24).An independent cohort of 64 patients with locally advancedbreast cancer who had been treated with neoadjuvant taxane–

JNK/MKP-1 Interplay Determines Resistance in Breast Cancer

www.aacrjournals.org Mol Cancer Ther; 15(11) November 2016 2781




based chemotherapy was also included in the study. Pretreat-ment tumor specimens were histologically evaluated. For allcases, clinical data were collected from patients' medicalrecords by oncologists. The ethical committees and Institution-al Review Boards of the participating hospitals approved theproject.

Clinical tumor response to primary chemotherapy was evalu-ated according to the International Union Against Cancer Criteria(25). Clinical complete response (cCR) was defined as the dis-appearance of all detectable malignant disease within the breastby physical examination. A reduction greater than 50% in theproduct of the two maximum perpendicular diameters of thetumor was classified as clinical partial response (cPR). Clinicalprogressive disease (cPD) was considered as an increase of at least25%. Clinical stable disease (cSD) was defined as situations inwhich clinical breast cancer response did not meet the criteria forcCR, cPR, or cPD. Postchemotherapy specimens were evaluatedfor pathologic response. A pathologic complete response (pCR)was defined as no histologic evidence of invasive disease in thetumor specimen (26).

ImmunohistochemistryImmunostainings were performed on tissue sections (3 mm)

obtained from FFPE tumors as previously described (15). Allstainings were performed in a Dako Autostainer. Sections incu-batedwithnormal nonimmunized rabbit immunoglobulinswereused as negative controls. Sections of breast tumor with knownexpression of targets were used as positive controls. Antibodysensitivity was calculated in a range of crescent dilutions ofprimary antibody (MKP-1, 1:50-1:200; p-JNK, 1:10-1:200; JNK,1:100-1:1,000). Specificity was confirmed in a set of paired freshfrozen and FFPE samples processed by WB and IHC. Antigenpreservation in tissues was confirmed by expression of phospho-tyrosines using a monoclonal antibody to tyrosine-phosphory-lated proteins (clone 4G10, 1:500; Millipore). High proliferationin breast cancer based on Ki67 labeling by IHC was definedfollowing the 13th St. Gallen International Breast Cancer Con-ference (2013) criteria based on a proliferation threshold �20%(5). Only the membrane of epithelial cells, but not stromal cells,was evaluated for MKP-1, p-JNK, and JNK. Expression blinded toclinical data was evaluated by two pathologists (F. Rojo and S.Zazo). A semiquantitative histoscore (Hscore) was calculated byestimating the percentage of tumor cells positively stained withlow, medium, or high staining intensity. The formula used wasHscore¼ (low%)�1þ (medium%)� 2þ (high%)� 3, and theresults ranged from 0 to 300.

Statistical analysisStatistical analyses were performed using the software SPSS

20 (SPSS Inc.). For in vitro studies, we included at least inde-pendent triplicates for all the cases. Receiver operating curve(ROC) analysis was used to determine the optimal cutoff pointbased on progression end point for MKP-1, p-JNK, and JNKexpression as previously described (27). Overall survival (OS)was defined as the time from diagnosis to the date of deathfrom any cause or last follow-up. Disease-free survival (DFS)was defined as the time from diagnosis until the first event, inwhich relapse at any location, death, or end of follow-up wereconsidered events. Survivals were analyzed by the Kaplan–Meier method using the log-rank test. Multivariate analyseswere carried out using the Cox proportional hazards model.

Analysis of experimental conditions was done by paired t test.All statistical tests were conducted at the two-sided 0.05 level ofsignificance. This work was carried out in accordance withReporting Recommendations for Tumor Marker PrognosticStudies (REMARK) guidelines (28).

ResultsChemotherapy treatments activate MAPK through MKP-1 inbreast cancer cell lines

Preliminary cell viability assays were performed in MDA-MB-231 and BT-474 breast cancer cell lines to evaluate the effects ofdocetaxel and doxorubicin on MAPK activation (IC50 of 48.3nmol/L and 17.3 mmol/L were calculated, respectively, data notshown). Gene expression changes associated with the drug treat-ments were revealed, after BT-474 cells were exposed to docetaxelfor 4 hours, by differentially expressed levels in more than 300genes as comparedwith nontreated cells (GEOaccession number:#16789213; Fig. 1A). Noticeably, several MKPs (such as MKP-1,MKP-2, andMKP-3)were among the 25 topdownregulated genes.Similar results were found when MDA-MB-231 cells were treatedwith doxorubicin (Fig. 1A). MKP-1 downregulation resultingfrom chemotherapy treatment was confirmed by qPCR in bothcell lines (Fig. 1B): in both cases, the effect of docetaxel wassignificant only at concentrations double of IC50; doxorubicin, onthe other hand, repressed the levels of MKP-1 even at concentra-tions below its IC50, with a more drastic effect when a concen-tration double of IC50 was used.

We postulated that this inhibitory effect of docetaxel anddoxorubicin in several MKPs was channeled by differentMAPKs. Therefore, we treated MDA-MB-231 and BT-474 cellswith docetaxel 50 nmol/L and doxorubicin 10 mmol/L for 24hours to further quantify the phosphorylation status of MAPKs.As expected, WB analyses revealed higher phosphorylationlevels of JNK1/2 and ERK1/2 in both breast cancer cell lines(Fig. 1C). In addition, we confirmed the inhibition of MKP-1after doxorubicin treatment in both cell lines. The apparent lackof modulation of MKP-1 protein expression by docetaxel wasdiscarded when the effect was measured at larger times (i.e., by48 hours of docetaxel treatment, the decrease of MKP-1 signalwas evident, and by 72 hours, there was no detectablesignal; Fig. 1D; Supplementary Fig. S1). Collectively, these datasuggest that MKP-1 could mediate the responses of MAPKs tochemotherapy in breast cancer cells.

MKP-1 overexpression improves cell survival against docetaxeland doxorubicin in breast cancer cell lines

By transfecting both cell lines with a plasmid constructioncontaining the MKP-1 clone cDNA, we observed a sustainedincrease in MKP-1 transcript levels. These levels were practicallyunaltered despite docetaxel or doxorubicin treatment (Fig. 2A). Atthe protein level, however, MKP-1 expression was abolished bydoxorubicin in both cell lines (both endogenous and ectopicexpression), indicating that the drug triggers a posttranscriptionalmechanism on the cells. Docetaxel, on the other hand, did notshow any effect on MKP-1 protein levels (Fig. 2B).

The overexpression of MKP-1 provided the breast cancer celllines with a higher resistance to docetaxel or doxorubicin, asdemonstrated by a significantly increased capacity for cell growth(Fig. 2C). The ability of transfected cells to resist exposure tochemotherapy was quantified from crystal violet images as the

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Mol Cancer Ther; 15(11) November 2016 Molecular Cancer Therapeutics2782




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MKP-1 is involved in the response of breast cancer cells to docetaxel and doxorubicin. A,microarray gene expression profiles of MDA-MB-231 and BT-474 cells afterdoxorubicin and docetaxel treatment for 24 hours, respectively, showing the top 25 down- and upregulated genes (as compared with nontreated controlcells). MKP-1, MKP-2, and MKP-3 (labeled with their official symbols as DUSP1, DUSP4, and DUSP6, respectively) were among those. Green, underexpression; red,overexpression. B, gene expression analysis by real-time qPCR of MKP-1 in MDA-MB-231 and BT-474 cells after docetaxel (D, nmol/L) or doxorubicin (X, mmol/L)treatments for 24 hours. C, nontreated control cells. Results are expressed as RQ (reference gene: ATP5E). Experiments were repeated at least three times.� , P < 0.05; �� , P < 0.01. C, docetaxel and doxorubicin regulate the activation of JNK1/2 and ERK1/2 and the expression of MKP-1 in breast cancer cells. WB analysisshowing the molecular effects induced after docetaxel (50 nmol/L) and doxorubicin (10 mmol/L) treatment for 24 hours in MDA-MB-231 and BT-474 cells.D, docetaxel causes decreased MKP-1 protein levels and activation of JNK1/2 and ERK1/2 in a time-dependent manner. Details as in C.






relative cell area in colorimetric growth assays (SupplementaryFig. S2). Cell viability usingMTS assays confirmed higher viabilityvalues in transfected cells than in control cells, both in MDA-MB-

231 and BT-474 cells after 24 hours of docetaxel (50 nmol/L) ordoxorubicin (10 mmol/L), with statistical significance reached inall conditions (Fig. 2D). Finally, MKP-1 overexpression elicited a

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MKP-1 induced overexpression inbreast cancer cells and its effect onchemoresistance. A, gene expressionanalysis of MKP-1 by qPCR in MDA-MB-231 and BT-474 cells after chemotherapytreatment. The two first bars in eachpanel show the increase in MKP-1 mRNAexpression following the transfectionwith the MKP-1 plasmid. The followingbars display the effect of docetaxel (D,nmol/L) and doxorubicin (X, mmol/L)treatments. Note that in these cases, thesample "HA-MKP-1" was used as thecalibrator (bar "C"). Data are expressedas RQ with respect to the ATP5Ereference gene. Bars in dark gray colorrepresent cell lines transfected with anempty vector (pCMV-HA-;); in light gray,transfectionwith theMKP-1 gene (pCMV-HA-MKP-1). Other details as in Fig. 1.B, WB analysis showing MKP-1overexpression after plasmidictransfection and chemotherapytreatment in MDA-MB-231 and BT-474cells. The analysis revealed thedeleterious effect of doxorubicin onMKP-1 overexpression, suggesting aposttranscriptional effect of doxorubicinon MKP-1 protein levels. Docetaxel, onthe contrary, did not alter MKP-1 proteinlevels. Note that MKP-1 appears as adouble band, as a result of theconcomitant endogenous (wild-typeprotein, MW: �40 kDa) and ectopicexpression (protein plus HA tag, MW:�50 kDa) of the protein species. C,relative cell area analysis from crystalviolet colorimetric growth assayafter MKP-1 overexpression andchemotherapy treatment in MDA-MB-231and BT-474 cells. D, relative cell viabilityanalysis from MTS assay. E, apoptosisfold-change from Annexin V andpropidium iodide staining after MKP-1overexpression and chemotherapytreatment in MDA-MB-231 and BT-474cells. Other details as in previous figures.� , P < 0.05; ��, P < 0.01.

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noticeable strong survival increase in breast cancer cells.Changes in apoptotic ratios increased cell survival by an orderof magnitude (Fig. 2E), and although docetaxel and doxoru-bicin treatments were effective in bringing about a significantreduction in cell viability, they did not reach the levels ofparental cells.

JNK inactivation byMKP-1 overexpression reduces apoptosis inbreast cancer cell lines

Given that alteration in MKP-1 protein-expression level wasapparently conditioning the viability and apoptotic behavior ofbreast cancer lines, and considering JNK1/2 as one of the mainproapoptotic regulators of the cell, we evaluated the activationlevels of JNK1/2 in MDA-MB-231 and BT-474 cells transientlyoverexpressing MKP-1. WB analysis showed that MKP-1 over-expression was able to dephosphorylate JNK1/2 in both celllines (Fig. 3A). To reveal the key role of JNK1/2 in the regu-lation of MKP-1–mediated response to docetaxel or doxorubi-cin treatment, we assessed the response of the transfected cellsin the presence of well-known JNK1/2 modulators: anisomycinas an activator and SP6000125 as an inhibitor agent. Accord-ingly, the effect of doxorubicin treatment in MKP-1–overex-pressing cells was highly marked in combination withthe addition of anisomycin, revealing both an increase inJNK1/2 activation (phosphorylation) and the disappearanceof the MKP-1 endogenous protein band (but not the ectopicband). The effect was more evident in MDA-MB-231 than in BT-474 cells. This point is in agreement with the rise of apoptoticrates in the presence of anisomycin. On the other hand, theoverexpression of MKP-1 cleared the phosphorylation signalingmediated by JNK1/2 when the cells were exposed to the JNK1/2inhibitor, and this effect could not be reverted by docetaxel ordoxorubicin (Fig. 3A). In general, the effect of the overexpres-sion of MKP-1 exceeded the modulation of MAPKs by activatoror inhibitory agents.

Coinciding with protein-expression alterations, MKP-1 over-expression resulted in increased viability of the transfected cellsafter docetaxel or doxorubicin treatments, despite the presenceof a JNK1/2 modulator (Fig. 3B). As expected, the net balance ofcell proliferation was lower in anisomycin-pretreated cells, andabove the balance for non-pretreated control cells in theSP6000125-treated cells. At the same time, the MKP-1–trans-fected cells showed higher viability rates than the cells with theempty vector. This double combination of MKP-1 overexpres-sion and JNK1/2 chemical premodulation when cells are trea-ted with either docetaxel or doxorubicin led us to the conclu-sion that the cellular response to chemotherapeutic treatmentsis mediated by JNK1/2, and that the response is conditioned bythe expression level of MKP-1. Similar conclusions were drawnwhen apoptosis was measured in cells under different condi-tions (Fig. 3C): MKP-1 overexpression resulted in partial inhi-bition of JNK1/2-induced apoptosis after chemotherapeutictreatment. The proapoptotic effect of docetaxel or doxorubicintreatments was significantly repressed by the expression of extraquantities of MKP-1, even in the presence of anisomycin, whenthe induction of JNK1/2 should be activating the proapoptoticpathway. On the contrary, the addition of SP6000125 prior tothe treatment nearly abolished the apoptotic response to doc-etaxel in both cell lines—a fact not altogether striking fordoxorubicin—confirming the key role of JNK/MKP-1 interplayin the cellular response to chemotherapy.

Prevalence of MKP-1 and activated JNK expression in humanbreast cancer

In order to understand the clinical implications of thesefindings, we investigated the prevalence and clinical signifi-cance of MKP-1 overexpression and its relation with JNK1/2activation. To do this, we quantified MKP-1 and p-JNK1/2expression in a cohort of 350 tumors obtained from patientswith early breast cancer treated with adjuvant anthracycline–based chemotherapy. Patient characteristics are shown inSupplementary Table S1. The IHC analysis of MKP-1 andp-JNK1/2 (Fig. 4A) showed that MKP-1 and p-JNK1/2 werediffusely distributed throughout the tumor, with primaryexpression located in the nucleus of tumor cells. Faint levelsof MKP-1 and moderate signal for p-JNK1/2 were detected innormal breast epithelium and stromal cells (SupplementaryFig. S3).

High MKP-1 levels were detected in 31% of the samples. Theelevated expression of MKP-1 was associated with the size of thetumors (P ¼ 0.013) and with relapse (P < 0.001), but was notdependent on the molecular subtype of the tumors. Amongtumors with high MKP-1 expression, 80% of the samples pre-sented low levels of p-JNK1/2. It was therefore not a surprise thatJNK1/2 inhibition was associated with the same parameters ashigh MKP-1 expression (tumor size and relapse), the clinicalbehavior reinforcing our previous understanding about themolecular relationship between MKP-1 and JNK1/2. The associ-ation between MKP-1 and p-JNK1/2 expression levels, as well asthe molecular and clinical parameters of this series, is includedin Table 1.

JNK activation and elevated MKP-1 expression determinebenefit of chemotherapy in human breast cancer

Complete data from clinical follow-up were available forall the 350 patients included in the study. Of relevance, MKP-1 overexpression was found in those patients who relapsed(P < 0.001). Moreover, the subgroup of patients with MKP-1overexpression showed substantially shorter OS (P < 0.001)and DFS (P < 0.001); among these patients, those withp-JNK1/2 inhibition presented the worst survival prognosis(Fig. 4B). Multivariate Cox analysis revealed that the combi-nation of MKP-1(þ) and p-JNK1/2(–) determinations pro-vided an independent marker for adverse outcome associatedwith OS [HR, 26.1; 95% confidence interval (CI), 10.1–67.4;P < 0.001; Table 2] and DFS (HR, 33.4; 95% CI, 14.8–75.4;P < 0.001; Supplementary Table S2) in early breast cancer.

JNK activation and high MKP-1 expression determine responseto docetaxel in human breast cancer patients

In order to provide clinical evidence to prove that MKP-1overexpression determines docetaxel resistance, we analyzedMKP-1 and p-JNK1/2 expression in an independent set of 64patients with locally advanced breast cancer who receivedneoadjuvant taxane–based chemotherapy. Patient characteris-tics are shown in Supplementary Table S3. The mean time fromdiagnosis to the beginning of chemotherapy was 21.3 days(range, 1–48 days). During this period, patients underwentstandard clinical and radiological tumor staging. Patientsreceived a median of four cycles of chemotherapy (range, 2–6 cycles). After recovering from the effects of the chemotherapy,the patients underwent surgery. The mean time between the lastdose of chemotherapy and acquisition of the postchemotherapy






specimen from surgery was 30.3 days (range, 8–59 days).Almost 30% of patients achieved a complete pathologicresponse (as analyzed in the surgical specimen) according tothe histopathologic evaluation. Interestingly, we observed thatMKP-1 and p-JNK1/2 expression correlated with pathologicresponse (P ¼ 0.008; Supplementary Table S4).

DiscussionMKP-1has longbeen reported to act as anoncoprotein inbreast

cancer progression, also inducing antitumor response to severalchemotherapeutic drugs (13, 15). Its capability to dephosphor-ylate p38, JNK, and ERK1/2—specifically in this order of affinity(29)—has been proven to be context- and stimulus-dependent.

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Figure 3.

Effects of modulation of JNK1/2 expression (induction by anisomycin; inhibition by SP6000125) on the response of both parental and MKP-1–overexpressingbreast cancer cells to chemotherapy treatment. A, WB analysis of MDA-MB-231 and BT-474 cells confirmed that MKP-1 overexpression largely inhibited theactivation of JNK1/2 and that doxorubicin treatment (but not docetaxel) triggered an increase in JNK1/2 activation. Other details as in previous figures.B, cell viabilityanalysis from MTS assays after MKP-1 overexpression and treatments with JNK1/2 modulators and chemotherapy. C, apoptosis fold-change from AnnexinV and propidium iodide staining after MKP-1 overexpression and treatments with JNK1/2 modulators and chemotherapy. Other details as in previous figures.� , P < 0.05; �� , P < 0.01.

Rinc�on et al.





Conversely, it is well known that, under specific circumstances,MAPKs have the ability to control MKP-1 expression throughcomplex regulatory loops (30, 31), although the molecularmechanisms regulating these routes merit further clarification.

In a previous work, we revealed that MKP-1 is overexpressedduring the malignant transformation of the breast and indepen-dently predicts poor prognosis. Furthermore, we demonstratedthat MKP-1 is repressed by doxorubicin in many human breastcancers (15). In the present study, we demonstrate that MKP-1overexpression can be a crucial event in breast cancer, as breastcancer cells overexpressing MKP-1 increase their proliferationrate and acquire the capability to inhibit apoptosis activation,even following doxorubicin or docetaxel treatments. In addi-tion, we prove that JNK1/2 dephosphorylation is a leading

molecular event that correlates with MKP-1 expression topromote tumor-cell survival after chemotherapy treatment.MDA-MB-231 and BT-474 breast cancer cell lines overexpres-sing MKP-1 withstand doxorubicin and docetaxel treatments(e.g., enhanced cell proliferation, improved cell viability,reduced apoptotic rates) by dephosphorylating JNK1/2. Onthe contrary, enforced JNK1/2 activation (phosphorylation) byanisomycin increases proapoptotic signals after drug additionto almost parental levels, even in the presence of high MKP-1protein abundance.

From a clinical perspective, we report here that MKP-1 over-expression is a crucial event in almost a third of breast cancerpatients; further, we define JNK1/2 dephosphorylation as awidespread molecular event in this subset (80% of MKP-1–

B

Overall survival (y)

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Figure 4.

JNK activation and MKP-1 expression inhuman breast cancer determine benefit ofchemotherapy. A, IHC detection of MKP-1 andp-JNK1/2 showing positive and negativestaining in four representative tumor samples.The line shows 30 mm. Magnification,�200. B,Kaplan–Meier analyses of OS and DFS in acohort of 350 breast cancer patients.






overexpressing tumors). Moreover, in our series, patients withMKP-1 overexpression showed significantly worse outcome,and multivariate analysis suggests that MKP-1 overexpressionhas an independent prognostic value for OS and DFS inadjuvant anthracycline–based chemotherapy. In particular, thedephosphorylation of JNK1/2 appears to be a significantmolecular mechanism correlated to MKP-1–induced resistanceto the cytotoxic treatment in those tumors. Furthermore, weproved, in an independent cohort of samples with locallyadvanced breast cancer, that patients with MKP-1 overexpres-sion and JNK1/2 inhibition are significantly more resistant toneoadjuvant taxane–based chemotherapy. The IHC determina-tions of MKP-1 and p-JNK1/2 were associated with pathologicresponse in patients treated in neoadjuvancy, suggesting a keyrole of the MKP-1 / p-JNK1/2 interplay in the acquisition ofmalignant traits. Consequently, these results suggest that thecombination of MKP-1 overexpression and JNK1/2 inhibitionis a common and relevant molecular event with high clinicalimportance in breast cancer, as it defines a subset of tumorswith predicted lack of clinical success for some of the usualchemotherapeutic regimens. Finally, in our cohort of patients,MKP-1 overexpression did not correlate with biological subtype(Table 1), which would imply that the consequences of MKP-1overexpression may be as heterogeneous as the breast cancerheterogeneity itself.

Although the link between MKP-1 and chemoresistance hasbeen previously reported in an in vitro cellular model in breastcancer (13), no association with clinical findings had beendescribed to date. Our results confirmed the ability of MKP-1overexpression to inhibit JNK1/2 activation in tumor cell lines.Furthermore, we describe the clinical significance this molecularevent may have in a particular subset of breast tumors, where theoverexpression of MKP-1 would hinder the effect of adjuvantchemotherapy and enable tumor relapse.

In our subset of early breast cancer samples, most of the MKP-1–overexpressing tumors showed low levels of p-JNK1/2. How-ever, approximately 20% of these samples presented p-JNK1/2overexpression. Though it is widely accepted that elevated MKP-1expression is linked to dephosphorylation of JNK1/2, there arereports in the literature elucidating the contribution of JNK1/2phosphorylation to tumor progression in specific contexts: Forinstance, JNK pathway activation was linked with the upregula-tion of Ras in certain human tumors, including HER2-positivebreast cancer (32); casein kinase 1 epsilon–mutant breast cancerhas been reported to lead to the activation of the Wnt/Rac1/JNK/AP1 pathway instead of canonical Wnt/b-catenin, thus mediatinghigher invasion ability and aggressiveness of breast cancer cells(33); IL33 provoked epithelial cell transformation and breasttumorigenesis by activation of MEK-ERK, JNK-cJun, and STAT3through the IL33/ST2/COT cascade (34). In summary, the

Table 1. Association of MKP-1 and p-JNK expression with molecular and clinical parameters in 350 breast cancer patients

Number of samples MKP-1(�), n (%) MKP-1(þ)/pJNK(þ), n (%) MKP-1(þ)/pJNK(�), n (%) P

MKP-1/p-JNK expression 350 241 (68.9) 22 (6.3) 87 (24.9)T 350 241 22 87 0.0131 189 143 (59.3) 9 (40.9) 37 (42.5)2 126 81 (33.6) 10 (45.5) 35 (40.2)3 33 17 (7.1) 3 (13.6) 13 (14.9)4 2 0 (0.0) 0 (0.0) 2 (2.3)

N 350 241 22 87 0.1470 203 149 (61.8) 10 (45.5) 44 (50.6)1 87 57 (23.7) 8 (36.4) 22 (25.3)2 38 25 (10.4) 2 (9.1) 11 (12.6)3 22 10 (4.1) 2 (9.1) 10 (11.5)

Grade 350 241 22 87 0.8801 52 35 (14.5) 2 (9.1) 15 (17.2)2 163 111 (46.1) 11 (50.0) 41 (47.1)3 135 95 (39.4) 9 (40.9) 31 (35.6)

ER 350 241 22 87 0.911Negative 101 68 (28.2) 7 (31.8) 26 (29.9)Positive 249 173 (71.8) 15 (68.2) 61 (70.1)

PR 350 241 22 87 0.767Negative 134 92 (38.2) 7 (31.8) 35 (40.2)Positive 216 149 (61.8) 15 (68.2) 52 (59.8)

HER2 350 241 22 87 0.919Negative 271 188 (78.0) 17 (77.3) 66 (75.9)Positive 79 53 (22.0) 5 (22.7) 21 (24.1)

Molecular subtype (St. Gallen) 350 241 22 87 0.400Luminal A 162 119 (49.4) 6 (27.3) 37 (42.5)Luminal B HER2– 42 25 (10.4) 6 (27.3) 11 (12.6)Luminal B HER2þ 53 35 (14.5) 4 (18.2) 14 (16.1)HER2 26 18 (7.4) 1 (4.5) 7 (8.0)Triple-negative 67 44 (18.3) 5 (22.7) 18 (20.7)

Relapse 350 241 22 87 <0.001No 277 234 (97.1) 9 (40.9) 34 (39.1)Yes 73 7 (2.9) 13 (59.1) 53 (60.9)

Ki-67 350 241 22 87 0.140Low 241 169 (70.1) 11 (50.0) 61 (70.1)High 109 72 (29.9) 11 (50.0) 26 (29.9)

Rinc�on et al.





different isoforms of JNK can exert anti- and protumor modula-tions in different cell types and stages of cancer, including breasttumors, as has been reviewed elsewhere (35).

Despite severalmolecular issues yet to be clarified regarding themolecular interplay of MKP-1 in breast cancer, it is clear that theoverexpression of MKP-1 is a key molecular event that should beconsidered as a potential predictive biomarker in breast cancer.This alteration accompanying JNK1/2 dephosphorylation needsfurther study in order to understand the breast cancer prosurvivalmechanisms associated to them. Our clinical results revealed ahigh prevalence of this breast cancer subtype (MKP-1 overexpres-sion/JNK1/2 dephosphorylation) as nearly 1 of 4 breast cancerpatients with this molecular profile would not benefit fromconventional adjuvant chemotherapy treatment. What is more,most of these patients may suffer relapse after treatment. There-fore, the incorporation of MKP-1 determination to the screeningpanel of prognostic and predictive biomarkers in breast cancerwould facilitate the management of such patients, aiding in thedecision on which therapeutic regimens can best improve theirsurvival.

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

Authors' ContributionsConception and design: R. Rinc�on, R. Perona, J. Albanell, J. Madoz-G�urpide,F. Rojo

Development of methodology: R. Rinc�on, S. Zazo, C. Chamizo, R. Manso,P. Gonz�alez-Alonso, I. Crist�obal, J. Albanell, A. Rovira, J. Madoz-G�urpide,F. RojoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R. Rinc�on, S. Zazo, R. Manso, P. Gonz�alez-Alonso,C. Ca~nadas, A. Lluch, P. Eroles, J. García-Foncillas, F. RojoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R. Rinc�on, S. Zazo, R. Manso, P. Gonz�alez-Alonso,P. Eroles, J. García-Foncillas, J. Madoz-G�urpide, F. RojoWriting, review, and/or revision of the manuscript: R. Rinc�on, I. Crist�obal,P. Eroles, J. García-Foncillas, J. Albanell, A. Rovira, J. Madoz-G�urpide, F. RojoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): R. Rinc�on, E. Martín-Aparicio, J. Albanell,J. Madoz-G�urpide, F. RojoStudy supervision: I. Crist�obal, A. Lluch, J. Madoz-G�urpide, F. Rojo

AcknowledgmentsWe thank Oliver Shaw for linguistic correction of the article.

Grant SupportThe current work was supported by grants from the Spanish Ministry of

Economy and Competitiveness (MINECO; AES Program, grants PI12/01552;PI12/00680; PI12/01421); the Ministry of Health (Cancer Network); theCommunity of Madrid (S2010/BMD-2344 grant); and the Government ofCatalonia (2014/SGR/740 grant). The biobanks are funded by grants from theMINECO(Institute ofHealthCarlos III, RETICS BiobanksNetwork, with FEDERfunds: Fundaci�on Jim�enez Díaz Biobank, RD12/0036/0021; Parc de Salut MarBiobank, RD12/0036/0051; Valencia Clinic Hospital Biobank, RD12/0036/0070). S. Zazo and C. Chamizo are supported by grants from the Biobanks

Table 2. Univariate and multivariate Cox analyses in the cohort of 350 breast cancer patients (OS analysis)

Univariate OS analysis Multivariate OS analysisHR (95% CI) Significance HR (95% CI) Significance

T <0.001 0.104I 1.000 1.000II 2.729 (1.505–4.946) 1.474 (0.711–3.054)III 3.672 (1.636–8.246) 1.457 (0.494–4.299)IV 13.557 (3.966–46.341) 9.308 (1.546–56.036)

N <0.001 0.0130 1.000 1.0001 1.639 (0.854–3.143) 0.958 (0.443–2.071)2 2.236 (1.023–4.887) 0.499 (0.147–1.692)3 6.961 (3.537–13.700) 2.926 (1.251–6.844)

Grade 0.045 0.7961 1.000 1.0002 1.368 (0.559–3.347) 1.464 (0.480–4.466)3 2.377 (0.989–5.717) 1.426 (0.450–4.521)

ER 0.006 0.014Negative 1.000 1.000Positive 0.477 (0.285–0.797) 0.430 (0.219–0.845)

HER2 0.173Negative 1.000Positive 1.510 (0.851–2.678)

Ki-67 0.832Low 1.000High 0.937 (0.515–1.707)

Chemotherapy 0.580None 1.000Adjuvant 0.728 (0.362–1.463)Neoadjuvant 0.962 (0.373–2.481)

Hormone therapy 0.342No 1.000Yes 0.754 (0.425–1.338)

MKP-1/p-JNK1/2 <0.001 <0.001MKP-1(-) 1.000 1.000MKP-1(þ)/p-JNK1/2(þ) 4.923 (1.172–20.674) 4.518 (1.070–19.081)MKP-1(þ)/p-JNK1/2(-) 29.314 (11.523–74.575) 26.086 (10.103–67.353)






initiative. J. Albanell and F. Rojo are recipients of an intensification programISCIII/FEDER. R. Manso and P. Gonz�alez-Alonso are supported by Fundaci�onConchita R�abago de Jim�enez Díaz grants.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedNovember 16, 2015; revised July 29, 2016; accepted August 7, 2016;published OnlineFirst September 6, 2016.

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Rinc�on et al.




2016;15:2780-2790. Published OnlineFirst September 6, 2016.Mol Cancer Ther Raúl Rincón, Sandra Zazo, Cristina Chamizo, et al. Anthracyclines in Breast CancerKinase Phosphatase 1 Determines Resistance to Taxanes and c-Jun N-Terminal Kinase Inactivation by Mitogen-Activated Protein

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