TITLE PAGE Research article - Molecular Cancer...

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TITLE PAGE

Article type: Research article

Title: c-Jun N-terminal kinase inactivation by mitogen-activated protein kinase

phosphatase 1 determines resistance to taxanes and anthracyclines in breast cancer

Authors: Raúl Rincón1, Sandra Zazo1, Cristina Chamizo1, Rebeca Manso1, Paula

González-Alonso1, Ester Martín-Aparicio1, Ion Cristóbal2, Carmen Cañadas1, Rosario

Perona3, Ana Lluch4, Pilar Eroles4, Jesús García-Foncillas2, Joan Albanell5,6,7, Ana

Rovira5,6, Juan Madoz-Gúrpide1*, Federico Rojo1*

Affiliations: 1Pathology Department, IIS-Fundación Jiménez Díaz, UAM, E-28040

Madrid, Spain. 2Translational Oncology Division, Oncohealth Institute, Health

Research Institute FJD-UAM, University Hospital Fundación Jiménez Díaz, Madrid,

Spain. 3“Alberto Sols” Biomedical Research Institute CSIC-UAM, Madrid, Spain.

4Institute of Health Research INCLIVA, Valencia, Spain. 5Medical Oncology

Department, Hospital del Mar, Barcelona, Spain. 6Cancer Research Program, IMIM

(Hospital del Mar Research Institute), Barcelona, Spain. 7Universitat Pompeu Fabra,

Barcelona, Spain.

Corresponding authors: *Dr. Juan Madoz-Gúrpide, Ph.D., Pathology Department, IIS-

Fundación Jiménez Díaz, UAM, Avda. Reyes Católicos 2, E-28040 Madrid, Spain. E-

mail: [email protected]. Phone: +34-915504800.

*Dr. Federico Rojo, M.D. Ph.D, Pathology Department, University Hospital Fundación

Jiménez Díaz, Avda. Reyes Católicos 2, E-28040 Madrid, Spain. E-mail: [email protected].

Phone: +34-915504800.

Running title: JNK/MKP-1 interplay determines resistance in breast cancer.

Financial information: The authors declare no competing financial interests. The

present work was supported by grants from the Spanish Ministry of Economy and

on October 3, 2018. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 6, 2016; DOI: 10.1158/1535-7163.MCT-15-0920

http://mct.aacrjournals.org/

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Competitiveness (MINECO) (AES Program, grants PI12/01552; PI12/00680;

PI12/01421); the Ministry of Health (Cancer Network); the Community of Madrid

(S2010/BMD-2344 grant); and the Government of Catalonia (2014/SGR/740 grant).

The biobanks are funded by grants from the MINECO (Institute of Health Carlos III,

RETICS Biobanks Network, with FEDER funds: Fundación Jiménez Díaz Biobank,

RD12/0036/0021; Parc de Salut Mar Biobank, RD12/0036/0051; Valencia Clinic

Hospital Biobank, RD12/0036/0070). S. Zazo and C. Chamizo are supported by grants

from the Biobanks initiative. J. Albanell and F. Rojo are recipients of an intensification

program ISCIII/FEDER. R. Manso and P. González-Alonso are supported by Fundación

Conchita Rábago de Jiménez Díaz grants.




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ABSTRACT

Mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) is overexpressed

during malignant transformation of the breast in many patients, and it is usually

associated with chemoresistance through interference with JNK-driven apoptotic

pathways. Although the molecular settings of the mechanism have been documented,

details about the contribution of MKP-1 to the failure of chemotherapeutic interventions

are unclear. Transient overexpression of MKP-1 and treatment with JNK-modulating

agents in breast carcinoma cells confirmed the mediation of MKP-1 in the resistance to

taxanes and anthracyclines in breast cancer, through the inactivation of JNK1/2. We

next assessed MKP-1 expression and JNK1/2 phosphorylation status in a large cohort of

samples from 350 early breast-cancer patients treated with adjuvant anthracycline-based

chemotherapy. We detected that MKP-1 overexpression is a recurrent event

predominantly linked to dephosphorylation of JNK1/2 with an adverse impact on

relapse of the tumor and overall and disease-free survival. Moreover, MKP-1 and p-

JNK1/2 determinations in 64 locally advanced breast-cancer patients treated with

neoadjuvant taxane-based chemotherapy showed an inverse correlation between MKP-1

overexpression (together with JNK1/2 inhibition) and the pathological response of the

tumors. Our results emphasize the importance of MKP-1 as a potential predictive

biomarker for a subset of breast-cancer patients with worse outcome and less

susceptibility to treatment.




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INTRODUCTION

Improving breast-cancer therapy requires novel prognostic and predictive markers (1,

2). The prognostic labelling of this heterogeneous disease is still based on conventional

tumor–node–metastasis (TNM) staging and histopathological features (3, 4). While

estrogen receptor (ER)-dependent and progesterone-receptor (PR)-dependent tumors

and human epidermal growth factor receptor 2 (HER2)-positive subtypes have been

approved for non-cytotoxic regimens, the triple-negative breast-cancer subtype lacks a

therapy alternative to chemotherapy (5). Hence, the molecular dynamics that rule breast

cancer pathogenesis need to be elucidated to develop precise therapies to enhance

survival and overcome resistance to standard chemotherapy regimens (6, 7).

Mitogen-activated protein kinases (MAPKs) have been extensively described as

some of the key molecular events driving breast-cancer progression (8, 9). Part of

cancer-related MAPK regulation consists of a dual dephosphorylation performed by

MAPK phosphatases (MKPs) (8). Among them, MKP-1 plays a relevant role in

tumorigenesis, being able to dephosphorylate all MAPKs, with substrate preference for

p38 MAPK and c-Jun N-terminal kinase (JNK). The activation of the JNK pathway has

been linked to the apoptosis induced by several chemotherapeutic agents. In breast

cancer, JNK dephosphorylation has been correlated with cancer progression and tumor

survival against different stress conditions, such as chemotherapy or oxidative damage

(10-13). Further experimental research has related MKP-1 with tumor response to stress

in breast cancer. Of importance, a tuned MAPKs/MKPs balance regulates cellular

response to cancer therapy, as revealed by experimental evidence. For instance, in

breast-cancer cells, doxorubicin is able to activate JNK pathway to achieve its anti-

tumoral effect (14, 15). On the contrary, MKP-1 mediates different tumor responses to

anticancer therapy, depending on its activity or inactivity: MKP-1 activates anti-




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apoptotic pathways in response to proteasome inhibitors (16, 17) as well as anti-

proliferative activity to progesterone receptor in breast-cancer cells (18). Of relevance,

MKP-1 inhibition by small molecules enhanced the antitumoral effect of paclitaxel (19),

and transient expression 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 to taxanes- and anthracyclines-based therapies, as well as the

ability of MKP-1 to decrease JNK activation, we decided to explore the interplay of this

kinase/phosphatase pair in the resistance of breast cancer cells to docetaxel and

doxorubicin.

Previously, we showed that MKP-1 was overexpressed during the malignant

transformation of the breast, thus affecting MAPK expression, and its activation could

be inhibited by doxorubicin treatment (15). Nevertheless, we discovered that breast

tumors overexpressing MKP-1 did not show this MAPK alteration after this treatment

(15). In the present study, we confirm the importance of MKP-1 overexpression as a

negative prognostic marker of response to chemotherapy. Of importance, we

demonstrate that docetaxel and doxorubicin regulate ERK1/2 and JNK1/2 activation in

part through MKP-1 modulation. Further, MKP-1 overexpression dephosphorylates

JNK1/2 and results in higher cell growth and lower apoptotic rates in the tumor cells.




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METHODS

Cell cultures and reagents

MDA-MB-231 (ATCC HTB-26) and BT-474 (ATCC HTB-20) cell lines were

purchased from the American Type Culture Collection (ATCC, USA) and authenticated

according to the required standards (LGC Standards, Germany). Docetaxel,

doxorubicin, anisomycin, and SP6000125 were purchased from Sigma Aldrich. Human

MKP-1 cDNA was purchased from Open Biosystems and cloned into a pBluescriptR

vector (clone ID 4794895; Open Biosystems, Dharmacon, USA), digested with EcoRI

and KpnI enzymes, and inserted into a pCMV-HA plasmid. Transfections were carried

out using Lipofectamine 2000 (Life Technologies, USA) and following the

manufacturer’s indications.

Cell assays

Cell proliferation was measured in triplicate by MTS assay using the CellTiter 96

AQueous One Solution Cell Proliferation Assay (Promega, USA), following the

manufacturer’s indications. Cell growth was analyzed in triplicate by crystal violet

assay as previously reported (20). Apoptosis was measured using Annexin V FITC

Apoptosis Detection Kit I (BD Biosciences, USA) and quantified in a FACS CANTO II

cytometer (BD Biosciences).

Western blotting analysis

Western blotting (WB) analysis in protein extracts from cultured cells was done as

previously reported (21). Antibodies: anti–phospho-ERK1/2 (p-ERK1/2;

Thr202/Tyr204), anti-ERK1/2, and anti-JNK (Cell Signaling Technology, USA); anti-

active JNK pAb (p-JNK; Thr183/Tyr185) (Promega); anti-MKP-1 (Santa Cruz Biotech,

USA); anti-α-tubulin and anti-GAPDH (Sigma-Aldrich); and anti-Rabbit IgG (GE

Healthcare, USA).




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Microarray analysis

Total RNA from the cell lines was isolated using the RNeasy mini Kit (Qiagen, USA).

RNA purity and integrity were assessed both by spectrophotometry (NanoDrop ND-

2000, NanoDrop Technologies, USA) and electrophoresis (2100 Bioanalyzer, Agilent

Technologies, USA); for microarray experiments, the minimal requirements for RNA

purity were A260/280>2.0 and A260/230>1.4 and RIN>9.4. Microarray expression

profiles were obtained using the Affymetrix GeneChip Human Exon 1.0 ST Array

(Affymetrix Inc, USA). Following hybridization, the array was stained in the

Affymetrix GeneChip Fluidics Station 450 and scanned using a GeneChip Scanner 3000

7G.

Gene-expression profile analysis

Data were processed following the methodology previously described(22). Briefly, after

quality control of raw data, background was corrected, quantile-normalized, and

summarized to the gene level using the robust multi-chip average (RMA). Only those

transcripts with an intensity signal of more than 10% of all intensities of the mean of

studied groups and then over 50% of variance from total resting variance were

considered for further analysis. Linear Models for Microarray (LIMMA) were used for

detecting differentially expressed genes between conditions. Correction for multiple

comparisons was performed using the false discovery rate (FDR), and only genes with

an adjusted p-value <0.05 were selected as significant. For the purposes of functional

analysis, genes were selected to have an unadjusted p-value <0.05. Hierarchical cluster

analysis was also performed. All data analysis was performed in R with the packages

aroma.affymetrix, Biobase, LIMMA, and genefilter. Functional analysis was performed

with Ingenuity Pathway Analysis software (Ingenuity Systems, USA). All microarray

procedures were performed by the IMIM Microarray Core Facility (SAM).




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Quantitative real-time PCR

cDNA was produced using the Universal Transcriptor cDNA synthesis kit (Roche

Diagnostics, Switzerland) according to manufacturer’s recommendations. MKP-1 gene

expression levels were determined using a quantitative real-time PCR (qPCR) assay,

with ATP5E as a housekeeping gene. Primers were designed using the Lasergene Primer

design software (DNASTAR Inc., USA) and based on the following genomic

sequences: MKP-1 (NM_004417.3) and ATP5E (NM_006886.3). qPCRs were

performed using the LightCycler480 II system (Roche Applied Science, Switzerland)

for 45 cycles with the following set of primers: MKP-1, Fw, 5’-

GAGGCCATTGACTTCATAGAC-3’, and Rv, 5’-GTAAGCAAGGCAGATGGTG-3’;

ATP5E, Fw, 5’-GTAGCTGAGTCCAGCCTGTC-3’, and Rv, 5’-

GATCTGGGAGTATCGGATG-3’. Specific probes from the Universal Probe Library

(Roche Applied Science, USA) were selected. Relative gene-expression levels (RQ)

were calculated in accordance with the MIQE guidelines(23).

Patient samples

Three-hundred and fifty surgically resected specimens from primary breast tumors were

obtained from Parc de Salut Mar Biobank (MARBiobanc, Barcelona, Spain), Fundación

Jiménez Díaz Biobank (Madrid, Spain), and Valencia Clinic Hospital Biobank

(Valencia, Spain). Tumor specimens from formalin-fixed paraffin-embedded (FFPE)

blocks were retrospectively selected from consecutive breast-cancer patients diagnosed

between 1998 and 2000, following these criteria: infiltrating carcinomas, operable, no

neoadjuvant therapy, sufficient available tissue, and clinical follow-up. TNM staging

was classified using the American Joint Committee on Cancer (AJCC) staging system.

Histological grade was defined according to the Elston-Ellis modification of the Scarff-

Bloom-Richardson grading system (24). An independent cohort of 64 patients with




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locally advanced breast cancer who had been treated with neoadjuvant taxane-based

chemotherapy was also included in the study. Pre-treatment tumor specimens were

histologically evaluated. For all cases, clinical data were collected from patients’

medical records by oncologists. The ethical committees and institutional review boards

of the participating hospitals approved the project.

Clinical tumor response to primary chemotherapy was evaluated according to the

International Union Against Cancer Criteria (25). Clinical complete response (cCR) was

defined as the disappearance of all detectable malignant disease within the breast by

physical examination. A reduction greater than 50% in the product of the two maximum

perpendicular diameters of the tumor was classified as clinical partial response (cPR).

Clinical progressive disease (cPD) was considered as an increase of at least 25%.

Clinical stable disease (cSD) was defined as situations in which clinical breast cancer

response did not meet the criteria for cCR, cPR or cPD. Post-chemotherapy specimens

were evaluated for pathological response. A pathological complete response (pCR) was

defined as no histological evidence of invasive disease in the tumor specimen (26).

Immunohistochemistry

Immunostainings were performed on tissue sections (3μm) obtained from FFPE tumors

as previously described (15). All stainings were performed in a Dako Autostainer.

Sections incubated with normal non-immunized rabbit immunoglobulins were used as

negative controls. Sections of breast tumor with known expression of targets were used

as positive controls. Antibody sensitivity was calculated in a range of crescent dilutions

of primary antibody (MKP-1, 1:50-1:200; p-JNK, 1:10-1:200; JNK, 1:100-1:1000).

Specificity was confirmed in a set of paired fresh frozen and FFPE samples processed

by WB and IHC. Antigen preservation in tissues was confirmed by expression of

phospho-tyrosines using a monoclonal antibody to tyrosine-phosphorylated proteins




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(clone 4G10, 1:500, Millipore, USA). High proliferation in breast cancer based on Ki67

labelling by IHC was defined following the 13th St. Gallen International Breast Cancer

Conference (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 to clinical data was evaluated by two pathologists (FR and

SZ). A semiquantitative histoscore (Hscore) was calculated by estimating the

percentage of tumor cells positively stained with low, medium, or high staining

intensity. The formula used was Hscore = (low %) × 1 + (medium %) × 2 + (high %) ×

3, and the results ranged from 0 to 300.

Statistical analysis

Statistical analyses were performed using the software SPSS 20 (SPSS Inc, USA). For

in vitro studies we included at least independent triplicates for all the cases. Receiver

operating curve (ROC) analysis was used to determine the optimal cutoff point based on

progression end point for MKP-1, p-JNK and JNK expression as previously described

(27). Overall survival (OS) was defined as the time from diagnosis to the date of death

from any cause or last follow-up. Disease-free survival (DFS) was defined as the time

from diagnosis until the first event, in which relapse at any location, death, or end of

follow-up were considered events. Survivals were analyzed by the Kaplan–Meier

method using the log-rank test. Multivariate analyses were 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 of significance. This

work was carried out in accordance with Reporting Recommendations for Tumor

Marker Prognostic Studies (REMARK) guidelines (28).




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RESULTS

Chemotherapy treatments activate MAPK through MKP-1 in breast-cancer cell lines

Preliminary cell-viability assays were performed in MDA-MB-231 and BT-474 breast-

cancer cell lines to evaluate the effects of docetaxel and doxorubicin on MAPK

activation (IC50 of 48.3 nmol/L and 17.3 μmol/L were calculated, respectively. Data not

shown). Gene-expression changes associated to the drug treatments were revealed, after

BT-474 cells were exposed to docetaxel for 4 h, by differentially expressed levels in

more than 300 genes as compared to non-treated cells (GEO accession number:

#16789213) (Figure 1A). Noticeably, several MKPs (such as MKP-1, MKP-2, and

MKP-3) were among the 25 top downregulated genes. Similar results were found when

MDA-MB-231 cells were treated with doxorubicin (Figure 1A). MKP-1 downregulation

resulting from chemotherapy treatment was confirmed by qPCR in both cell lines

(Figure 1B): in both cases, the effect of docetaxel was significant only at concentrations

double of IC50; doxorubicin, on the other hand, repressed the levels of MKP-1 even at

concentrations below its IC50, with a more drastic effect when a concentration double of

IC50 was used.15

We postulated that this inhibitory effect of docetaxel and doxorubicin in several

MKPs was channeled by different MAPKs. Therefore, we treated MDA-MB-231 and

BT-474 cells with docetaxel 50 nmol/L and doxorubicin 10 μmol/L for 24 h to further

quantify the phosphorylation status of MAPKs. As expected, WB analyses revealed

higher phosphorylation levels of JNK1/2 and ERK1/2 in both breast-cancer cell lines

(Figure 1C). In addition, we confirmed the inhibition of MKP-1 after doxorubicin

treatment in both cell lines. The apparent lack of modulation of MKP-1 protein

expression by docetaxel was discarded when the effect was measured at larger times

(i.e., by 48 h of docetaxel treatment the decrease of MKP-1 signal was evident, and by




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72 h there was no detectable signal) (Figure 1D and Supplementary Figure S1).

Collectively, these data suggest that MKP-1 could mediate the responses of MAPKs to

chemotherapy in breast-cancer cells.

MKP-1 overexpression improves cell survival against docetaxel and doxorubicin in

breast-cancer cell lines

By transfecting both cell lines with a plasmid construction containing the MKP-1 clone

cDNA, we observed a sustained increase in MKP-1 transcript levels. These levels were

practically unaltered despite docetaxel or doxorubicin treatment (Figure 2A). At the

protein level, however, MKP-1 expression was abolished by doxorubicin in both cell

lines (both endogenous and ectopic expression), indicating that the drug triggers a

posttranscriptional mechanism on the cells. Docetaxel, on the other hand, did not show

any effect on MKP-1 protein levels (Figure 2B).

The overexpression of MKP-1 provided the breast-cancer cell lines with a higher

resistance to docetaxel or doxorubicin, as demonstrated by a significantly increased

capacity for cell growth (Figure 2C). The ability of transfected cells to resist exposure to

chemotherapy was quantified from crystal violet images as the relative cell area in

colorimetric growth assays (Supplementary Figure S2). Cell viability using MTS assays

confirmed higher viability values in transfected cells than in control cells, both in

MDA-MB-231 and BT-474 cells after 24 h of docetaxel 50 nmol/L or doxorubicin 10

μmol/L, with statistical significance reached in all conditions (Figure 2D). Finally,

MKP-1 overexpression elicited a noticeable strong survival increase in breast-cancer

cells. Changes in apoptotic ratios increased cell survival by an order of magnitude

(Figure 2E), and although docetaxel and doxorubicin treatments were effective in

bringing about a significant reduction in cell viability, they did not reach the levels of

parental cells.




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JNK inactivation by MKP-1 overexpression reduces apoptosis in breast-cancer cell

lines

Given that alteration in MKP-1 protein-expression level was apparently conditioning

the viability and apoptotic behavior of breast-cancer lines, and considering JNK1/2 as

one of the main pro-apoptotic regulators of the cell, we evaluated the activation levels

of JNK1/2 in MDA-MB-231 and BT-474 cells transiently overexpressing MKP-1. WB

analysis showed that MKP-1 overexpression was able to dephosphorylate JNK1/2 in

both cell lines (Figure 3A). To reveal the key role of JNK1/2 in the regulation of MKP-

1-mediated response to docetaxel or doxorubicin treatment, we assessed the response of

the transfected cells in the presence of well-known JNK1/2 modulators: anisomycin as

an activator and SP6000125 as an inhibitor agent. Accordingly, the effect of

doxorubicin treatment in MKP-1-overexpressing cells was highly marked in

combination with the addition of anisomycin, revealing both an increase in JNK1/2

activation (phosphorylation) as well as the disappearance of the MKP-1 endogenous

protein band (but not the ectopic band). The effect was more evident in MDA-MB-231

than in BT-474 cells. This point is in agreement with the rise of apoptotic rates in the

presence of anisomycin. On the other hand, the overexpression of MKP-1 cleared the

phosphorylation signaling mediated by JNK1/2 when the cells were exposed to the

JNK1/2 inhibitor, and this effect could not be reverted by docetaxel or doxorubicin

(Figure 3A). In general, the effect of the overexpression of MKP-1 exceeded the

modulation of MAPKs by activator or inhibitory agents.

Coinciding with protein-expression alterations, MKP-1-overexpression resulted in

increased viability of the transfected cells after docetaxel or doxorubicin treatments,

despite the presence of a JNK1/2 modulator (Figure 3B). As expected, the net balance

of cell proliferation was lower in anisomycin-pretreated cells, and above the balance for




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non-pre-treated control cells in the SP6000125-treated cells. At the same time, the

MKP-1-transfected cells showed higher viability rates than the cells with the empty

vector. This double combination of MKP-1 overexpression and JNK1/2 chemical pre-

modulation when cells are treated with either docetaxel or doxorubicin led us to the

conclusion that the cellular response to chemotherapeutic treatments is mediated by

JNK1/2, and that the response is conditioned by the expression level of MKP-1. Similar

conclusions were drawn when apoptosis was measured in cells under different

conditions (Figure 3C): MKP-1 overexpression resulted in partial inhibition of JNK1/2-

induced apoptosis after chemotherapeutic treatment. The pro-apoptotic effect of

docetaxel or doxorubicin treatments was significantly repressed by the expression of

extra quantities of MKP-1, even in the presence of anisomycin, when the induction of

JNK1/2 should be activating the pro-apoptotic pathway. On the contrary, the addition of

SP6000125 prior to the treatment nearly abolished the apoptotic response to docetaxel

in both cell lines—a fact not altogether striking for doxorubicin—confirming the key

role of JNK/MKP-1 interplay in the cellular response to chemotherapy.

Prevalence of MKP-1 and activated JNK expression in human breast cancer

In order to understand the clinical implications of these findings, we investigated the

prevalence and clinical significance of MKP-1 overexpression and its relation with

JNK1/2 activation. To do this, we quantified MKP-1 and p-JNK1/2 expression in a

cohort of 350 tumors obtained from patients with early breast cancer treated with

adjuvant anthracycline-based chemotherapy. Patient characteristics are shown in

Supplementary Table S1. The IHC analysis of MKP-1 and p-JNK1/2 (Figure 4A)

showed that MKP-1 and p-JNK1/2 were diffusely distributed throughout the tumor,

with primary expression located in the nucleus of tumor cells. Faint levels of MKP-1




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and moderate signal for p-JNK1/2 were detected in normal breast epithelium and

stromal cells (Supplementary Figure S3).

High MKP-1 levels were detected in 31% of the samples. The elevated expression

of MKP-1 was associated with the size of the tumors (P = 0.013) and with relapse (P <

0.001), but was not dependent on the molecular subtype of the tumors. Among tumors

with high MKP-1 expression, 80% of the samples presented low levels of p-JNK1/2. It

was therefore not a surprise that JNK1/2 inhibition was associated with the same

parameters as high MKP-1 expression (tumor size and relapse), the clinical behavior

reinforcing our previous understanding about the molecular relationship between MKP-

1 and JNK1/2. The association between MKP-1 and p-JNK1/2 expression levels, as

well as the molecular and clinical parameters of this series, are included in Table 1.

JNK activation and elevated MKP-1 expression determine benefit of chemotherapy in

human breast cancer

Complete data from clinical follow-up were available for all 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-1 overexpression

showed substantially shorter OS (P < 0.001) and DFS (P < 0.001); among these

patients, those with p-JNK1/2 inhibition presented the worst survival prognosis (Figure

4B). Multivariate Cox analysis revealed that the combination of MKP-1(+) and p-

JNK1/2(-) determinations provided an independent marker for adverse outcome

associated with OS (HR 26.1; 95% 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 response to docetaxel in human

breast-cancer patients




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In order to provide clinical evidence to prove that MKP-1 overexpression determines

docetaxel resistance, we analyzed MKP-1 and p-JNK1/2 expression in an independent

set of 64 patients with locally advanced breast cancer who received neoadjuvant taxane-

based chemotherapy. Patient characteristics are shown in Supplementary Table S3. The

mean time from diagnosis to the beginning of chemotherapy was 21.3 days (range 1–48

days). During this period, patients underwent standard clinical and radiological tumor

staging. Patients received 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 last dose of chemotherapy and acquisition of the post-

chemotherapy specimen from surgery was 30.3 days (range 8–59 days). Almost 30% of

patients achieved a complete pathological response (as analyzed in the surgical

specimen) according to the histopathological evaluation. Interestingly, we observed that

MKP-1 and p-JNK1/2 expression correlated with pathological response (P = 0.008)

(Supplementary Table S4).




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DISCUSSION

MKP-1 has long been reported to act as an oncoprotein in breast-cancer progression,

also inducing antitumor response to several chemotherapeutic drugs (13, 15). Its

capability to dephosphorylate p38, JNK, and ERK1/2—specifically in this order of

affinity (29) —has been proven to be context- and stimulus-dependent. Conversely, it is

well-known that, under specific circumstances, MAPKs have the ability to control

MKP-1 expression through complex regulatory loops (30, 31), although the molecular

mechanisms regulating these routes merit further clarification.

In a previous work, we revealed that MKP-1 is overexpressed during the

malignant transformation of the breast and independently predicts poor prognosis.

Furthermore, we demonstrated that MKP-1 is repressed by doxorubicin in many human

breast cancers (15). In the present study we demonstrate that MKP-1 overexpression can

be a crucial event in breast cancer, as breast-cancer cells overexpressing MKP-1

increase their proliferation rate and acquire the capability to inhibit apoptosis activation,

even following doxorubicin or docetaxel treatments. In addition, we prove that JNK1/2

dephosphorylation is a leading molecular event that correlates with MKP-1 expression

to promote tumor-cell survival after chemotherapy treatment. MDA-MB-231 and BT-

474 breast-cancer cell lines overexpressing MKP-1 withstand doxorubicin and

docetaxel treatments (e.g., enhanced cell proliferation, improved cell viability, reduced

apoptotic rates) by dephosphorylating JNK1/2. On the contrary, enforced JNK1/2

activation (phosphorylation) by anisomycin increases pro-apoptotic signals after drug

addition to almost parental levels, even in the presence of high MKP-1 protein

abundance.

From a clinical perspective, we report here that MKP-1 overexpression is a crucial

event in almost a third of breast-cancer patients; further, we define JNK1/2




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dephosphorylation as a widespread molecular event in this subset (80% of MKP-1-

overexpressing tumors). Moreover, in our series, patients with MKP-1 overexpression

showed significantly worse outcome, and multivariate analysis suggests that MKP-1

overexpression has an independent prognostic value for OS and DFS in adjuvant

anthracycline-based chemotherapy. In particular, the dephosphorylation of JNK1/2

appears to be a significant molecular mechanism correlated to MKP-1-induced

resistance to the cytotoxic treatment in those tumors. Furthermore, we proved, in an

independent cohort of samples with locally advanced breast cancer, that patients with

MKP-1 overexpression and JNK1/2 inhibition are significantly more resistant to

neoadjuvant taxane-based chemotherapy. The IHC determination of MKP-1 and p-

JNK1/2 were associated with pathological response in patients treated in neoadjuvancy,

suggesting a key role of the MKP-1 / p-JNK1/2 interplay in the acquisition of malignant

traits. Consequently, these results suggest that the combination of MKP-1

overexpression and JNK1/2 inhibition is a common and relevant molecular event with

high clinical importance in breast cancer, as it defines a subset of tumors with predicted

lack of clinical success for some of the usual chemotherapeutic 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-1 overexpression may be

as heterogeneous as the breast-cancer heterogeneity itself.

Although the link between MKP-1 and chemoresistance has been previously

reported in an in vitro cellular model in breast cancer (13), no association with clinical

findings had been described to date. Our results confirmed the ability of MKP-1

overexpression to inhibit JNK1/2 activation in tumor cell lines. Furthermore, we

describe that the clinical significance this molecular event may have in a particular




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subset of breast tumors, where the overexpression of MKP-1 would hinder the effect of

adjuvant chemotherapy 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. However, approximately 20% of these samples

presented p-JNK1/2 overexpression. Though it is widely accepted that elevated MKP-1

expression is linked to dephosphorylation of JNK1/2, there are reports in the literature

elucidating the contribution of JNK1/2 phosphorylation to tumor progression in specific

contexts: for instance, JNK pathway activation was linked with the up-regulation of Ras

in certain human tumors, including HER2-positive breast cancer (32); casein kinase 1

epsilon-mutant breast cancer has been reported to lead to the activation of the

Wnt/Rac1/JNK/AP1 pathway instead of canonical Wnt/β-catenin, thus mediating higher

invasion ability and aggressiveness of breast-cancer cells (33); interleukin-33 (IL-33)

provoked epithelial cell transformation and breast tumorigenesis by activation of MEK-

ERK, JNK-cJun, and STAT3 through the IL-33/ST2/COT cascade (34). In summary,

the different isoforms of JNK can exert anti- and pro-tumor modulations in different cell

types and stages of cancer, including breast tumors, as has been reviewed elsewhere

(35).

Despite several molecular issues yet to be clarified regarding the molecular

interplay of MKP-1 in breast cancer, it is clear that the overexpression of MKP-1 is a

key molecular event that should be considered as a potential predictive biomarker in

breast cancer. This alteration accompanying JNK1/2 dephosphorylation needs further

study in order to understand the breast cancer pro-survival mechanisms associated to

them. Our clinical results revealed a high prevalence of this breast-cancer subtype

(MKP-1 overexpression/JNK1/2 dephosphorylation) as nearly 1 of 4 breast-cancer

patients with this molecular profile would not benefit from conventional adjuvant




20

chemotherapy treatment. What is more, most of these patients may suffer relapse after

treatment. Therefore, the incorporation of MKP-1 determination to the screening panel

of prognostic and predictive biomarkers in breast cancer would facilitate the

management of such patients, aiding in the decision on which therapeutic regimens can

best improve their survival.




21

ACKNOWLEDGMENTS

We thank Oliver Shaw for linguistic correction of the manuscript.




22

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27

TABLES

Table 1. Association of MKP-1 and p-JNK expression with molecular and clinical

parameters in 350 breast-cancer patients.

no. samples

MKP-1(-) no. (%)

MKP-1(+) / pJNK(+)no. (%)

MKP-1(+) / pJNK(-) no. (%)

P value

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 0.400

Luminal 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 HER2+

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)

ER: estrogen receptor; PR: progesterone receptor.




28

Table 2. Univariate and multivariate Cox analyses in the cohort of 350 breast-cancer patients

(OS analysis).

Univariate OS analysis Multivariate OS analysis HR 95% CI Significance HR 95% CI Significance

Lower Upper Lower Upper T <0.001 0.104

I 1.000 1.000 II 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 1.000 1 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 1.000 2 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 1.000 Positive 0.477 0.285 0.797 0.430 0.219 0.845

HER2 0.173 Negative 1.000 Positive 1.510 0.851 2.678

Ki-67 0.832 Low 1.000 High 0.937 0.515 1.707

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

Hormone therapy 0.342 No 1.000 Yes 0.754 0.425 1.338

MKP-1 / p-JNK1/2 <0.001 <0.001MKP-1(-) 1.000 1.000 MKP-1(+) / p-JNK1/2(+)

4.923 1.172 20.674 4.518 1.070 19.081

MKP-1(+) / p-JNK1/2(-)

29.314 11.523 74.575 26.086 10.103 67.353




29

FIGURE LEGENDS

Figure 1. 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 after doxorubicin and docetaxel treatment for 24 h, respectively, showing the top

25 down- and up-regulated genes (as compared to non-treated control cells). MKP-1,

MKP-2, and MKP-3 (labeled with their Official Symbols as DUSP1, DUSP4, and

DUSP6, respectively) were among those. Green color indicates underexpression; red

color indicates 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, μmol/L) treatments for 24 h. C: non-treated 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 regulates the activation of JNK1/2 and

ERK1/2 and the expression of MKP-1 in breast-cancer cells. WB analysis showing the

molecular effects induced after docetaxel (50 nmol/L) and doxorubicin (10 μmol/L)

treatment for 24 h 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 Figure 1C.

Figure 2. MKP-1 induced overexpression in breast-cancer cells and its effect on

chemoresistance. A. Gene-expression analysis of MKP-1 by qPCR in MDA-MB-231

and BT-474 cells after chemotherapy treatment. The two first bars in each panel show

the increase in MKP-1 mRNA expression following the transfection with the MKP-1

plasmid. The following bars display the effect of docetaxel (D, nmol/L) and

doxorubicin (X, μmol/L) treatments. Note that in these cases the sample “HA-MKP-1”

was used as the calibrator (bar “C”). Data were expressed as RQ with respect to the

ATP5E reference gene. Bars in dark gray color represent cell lines transfected with an




30

empty vector (pCMV-HA-∅); in light gray, transfection with the MKP-1 gene (pCMV-

HA-MKP-1). Other details as in Figure 1. B. WB analysis showing MKP-1

overexpression after plasmidic transfection and chemotherapy treatment in MDA-MB-

231 and BT-474 cells. The analysis revealed the deleterious effect of doxorubicin on

MKP-1 overexpression, suggesting a posttranscriptional effect of doxorubicin on MKP-

1 protein levels. Docetaxel, on the contrary, did not alter MKP-1 protein levels. Note

that MKP-1 appears as a double band, as a result of the concomitant endogenous (wild-

type protein, MW: ~40 kDa) and ectopic expression (protein plus HA tag, MW: ~50

kDa) of the protein species. C. Relative cell area analysis from crystal violet

colorimetric growth assay after MKP-1 overexpression and chemotherapy treatment in

MDA-MB-231 and BT-474 cells. D. Relative cell-viability analysis from MTS assay. E.

Apoptosis fold-change from annexin V and propidium iodide staining after MKP-1

overexpression and chemotherapy treatment in MDA-MB-231 and BT-474 cells. Other

details as in previous figures.

Figure 3. Effects of modulation of JNK1/2 expression (induction by anisomycin;

inhibition by SP6000125) on the response of both parental and MKP-1-overexpressing

breast-cancer cells to chemotherapy treatment. A. WB analysis of MDA-MB-231 and

BT-474 cells confirmed that MKP-1 overexpression largely inhibited the activation 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-viability analysis from

MTS assays after MKP-1 overexpression and treatments with JNK1/2 modulators and

chemotherapy. C. Apoptosis fold-change from annexin V and propidium iodide staining

after MKP-1 overexpression and treatments with JNK1/2 modulators and

chemotherapy. Other details as in previous figures.




31

Figure 4. JNK activation and MKP-1 expression in human breast cancer determines

benefit of chemotherapy. A. IHC detection of MKP-1 and p-JNK1/2 showing positive

and negative staining in 4 representative tumor samples. The line shows 30 µm.

Magnification: ×200. B. Kaplan-Meier analyses of OS and DFS in a cohort of 350

breast-cancer patients.




Figure 1

A BT-474

docetaxel

logFC

MDA-MB-231

doxorubicin

logFC

B

0,0

0,5

1,0

1,5

C D10 D100 X5 X50

MK

P-1

ge

ne

ex

pre

ss

ion

leve

ls

MDA-MB-231

*

*

*

*

0,0

0,5

1,0

1,5

C D10 D100 X5 X50

MK

P-1

ge

ne

ex

pre

ss

ion

leve

ls

BT-474

* **

**

C D X

MDA-MB-231

MKP-1

p-JNK1/2

JNK1/2

p-ERK1/2

ERK1/2

GAPDH

BT-474 C C D X

D

MKP-1

p-JNK1/2

p-ERK1/2

α-Tubulin

0h 12h 24h 48h 72h

C C D C D C D C D




Figure 2

C MDA-MB-231

0

20

40

60

80

100

120

Rela

tive

cell

are

a (

%)

Control D10 D20 D100 X5 X10 X50

*

**

* *

*

BT-474

0

20

40

60

80

100

120

140

Control D10 D20 D100 X5 X10 X50

*

*

*

* *

*

D

0

20

40

60

80

100

120

Cell

via

bili

ty (

%)

Control D50 X10

* *

0

20

40

60

80

100

120

Control D50 X10

* **

E

Control D50 X10

0

5

10

15

20

25

*

0

5

10

15

20

25

Apop

tosis

fold

change

(%)

Control D50 X10

** * pCMV-HA-

pCMV-HA-MKP-1

pCMV-HA-

pCMV-HA-MKP-1

D 50

X 10

+ + + - - -

- - - + + +

- + - - + -

- - + - - +

+ + + - - -

- - - + + +

- + - - + -

- - + - - +

MKP-1

GAPDH

MDA-MB-231 BT-474

B

A M

KP

-1 m

RN

A e

xp

ress

ion

(rela

tive

un

its)

0

25

50

75

100MDA-MB-231

0.0

0.5

1.0

1.5

2.00

15

30

45

60BT-474

0.0

0.5

1.0

1.5

2.0




Figure 3

B

Cell

via

bili

ty (

%)

0

20

40

60

80

100

120

140

Rela

tive c

ell

via

bil

ity (

%)

0

20

40

60

80

100

120

140

Rela

tive c

ell

via

bil

ity (

%)

D50

X10

anisomycin

- - - - + + - - - - + + - -

- - - - - - + + - - - - + +

SP6000125

* **

* *

*

** *

* *

MD

A-M

B-2

31

BT

-474

pCMV-HA-

pCMV-HA-MKP-1

C - - + - - - + - - - + -

- - - + - - - + - - - +

Apop

tosis

fold

change

(%

)

0

5

10

15

20

250

5

10

15

20

25

anisomycin

SP6000125

D50 X10

**

*

*

MD

A-M

B-2

31

BT

-474

*

**

*

*

*

A

GAPDH

+ + + + + + +

- - - - - - -

- - + - - + -

- - - + - - +

- + + + - - -

- - - - + + +

- - - - - - -

+ + + + + + +

- - + - - + -

- - - + - - +

- + + + - - -

- - - - + + +

pCMV-HA-

pCMV-HA-MKP-1

docetaxel

doxorubicin

anisomycin

SP6000125

MD

A-M

B-2

31

MKP-1

p-JNK1/2

JNK1/2

BT

-47

4

GAPDH

MKP-1

JNK1/2

p-JNK1/2




B

Overall survival (y)

MKP-1 (-)

MKP-1 (+), p-JNK1/2 (+)

MKP-1 (+), p-JNK1/2 (-)

P < 0,001

Cum

ula

tive

surv

ival

Disease-free survival (y)

P < 0,001

MKP-1 (-)

MKP-1 (+), p-JNK1/2 (+)

MKP-1 (+), p-JNK1/2 (-)

A

MKP-1 (+)

MKP-1 (+)

pJNK1/2 (+)

MKP-1 (-)

MKP-1 (-)

pJNK1/2 (+)

pJNK1/2 (-)

pJNK1/2 (-)

Tu

mo

r #

1

Tu

mo

r #

3

Tu

mor

# 2

T

um

or

# 4

Figure 4 on October 3, 2018. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from



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