High ERK Protein Expression Levels Correlate with Shorter Survival in

Triple-Negative Breast Cancer Patients

Chandra Bartholomeusz, Ana M. Gonzalez-Angulo, Ping Liu, Naoki Hayashi, Ana
Lluch, Jaime Ferrer-Lozano and Gabriel N. Hortobágyi

The Oncologist 2012, 17:766-774.

doi: 10.1634/theoncologist.2011-0377 originally published online May 14, 2012

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Oncologist
The ®

Breast Cancer

High ERK Protein Expression Levels Correlate with Shorter Survival

in Triple-Negative Breast Cancer Patients
CHANDRA BARTHOLOMEUSZ,a,b ANA M. GONZALEZ-ANGULO,b,c PING LIU,d NAOKI HAYASHI,a,b
ANA LLUCH,e JAIME FERRER-LOZANO,e GABRIEL N. HORTOBÁGYIa,b
a
Breast Cancer Translational Research Laboratory and Departments of bBreast Medical Oncology, cSystems
Biology, and dBiostatistics, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA;
e

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Hematology-Oncology and Pathology, Hospital Clínico Universitario de Valencia, Valencia, Spain

Key Words. Triple-negative breast cancer • ERK • Survival • PEA-15 • RPPA

Disclosures: Gabriel N. Hortobágyi: Allergan, Genentech, Merck, Novartis, Sanofi (C/A); Novartis (RF). The other authors indicated no
financial relationships.

(C/A) Consulting/advisory relationship; (RF) Research funding; (E) Employment; (H) Honoraria received; (OI) Ownership interests; (IP)
Intellectual property rights/inventor/patent holder; (SAB) Scientific advisory board

ABSTRACT
The mitogen-activated protein kinase (MAPK) signaling characteristics and outcome. The median age of patients
pathway is known to be activated in triple-negative breast with TNBC was 55 years (range, 27– 86 years). Disease
cancer (TNBC). Extracellular signal–related kinase stage was I in 21%, II in 60%, and III in 20% of the pa-
(ERK), a member of the MAPK pathway, promotes cell tients. In a multivariate analysis, among patients with
proliferation, angiogenesis, cell differentiation, and cell TNBC, those with ERK-2– overexpressing tumors had a
survival. To assess the prognostic impact of ERK in TNBC lower overall survival rate than those with low ERK-2–
patients, relative quantities of ERK (ERK-2 and pMAPK) expressing tumors (hazard ratio [HR], 2.76; 95% confi-
and direct targets of the ERK pathway (MAPK/ERK ki- dence interval [CI], 1.19 – 6.41). However, high pMAPK
nase 1, phospho-enriched protein in astrocytes [PEA]-15, levels were associated with a significantly higher relapse-
phosphorylated (p)PEA-15, tuberous sclerosis protein 2, free survival rate (HR, 0.66; 95% CI, 0.46 – 0.95). In con-
p70S6 kinase, and p27) were measured using reverse-phase clusion, ERK-2 and pMAPK are valuable prognostic
protein arrays in tumor tissue from patients with TNBC markers in TNBC. Further studies are justified to eluci-
(n ! 97) and non-TNBC (n ! 223). Protein levels in pa- date ERK’s role in TNBC tumorigenicity and metastasis.
tients with TNBC were correlated with clinical and tumor The Oncologist 2012;17:766 –774

INTRODUCTION about 85% of all basal-like tumors [1]. Currently, TNBC is the
Breast tumors that are negative for estrogen receptor, proges- only major type of breast cancer for which no specific U.S.
terone receptor, and human epidermal growth factor receptor Food and Drug Administration–approved targeted therapy is
(HER)-2 on immunohistochemistry and/or fluorescence in situ available to improve patient outcomes [2].
hybridization studies are classified as triple-negative breast In TNBCs, the mitogen-activated protein kinase (MAPK)
cancer (TNBC). Of the four molecular subgroups of breast [3, 4] and Akt [5] pathways are known to be activated. Extra-
cancer—luminal, ErbB-2, normal, and basal-type— basal- cellular signal–related kinase (ERK), a member of the MAPK
type is the subgroup to which TNBCs most often belong. Al- pathway, plays an important role in cell proliferation and dif-
though TNBCs share many characteristics with basal-type ferentiation, promotes epithelial–mesenchymal transition, and
tumors, these two groups are not identical: TNBCs comprise facilitates cell migration because of its effects on cell–matrix

Correspondence: Chandra Bartholomeusz, M.D., Ph.D, Department of Breast Medical Oncology, Unit 1354, The University of Texas MD
Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA. Telephone: 713-745-1086; Fax: 713-745-9296; e-
mail: [email protected] Received November 2, 2011; accepted for publication March 21, 2012; first published online in The
Oncologist Express on May 14, 2012. ©AlphaMed Press 1083-7159/2012/$20.00/0 http://dx.doi.org/10.1634/theoncologist.2011-0377

The Oncologist 2012;17:766 –774 www.TheOncologist.com

Bartholomeusz, Gonzalez-Angulo, Liu et al. 767

contacts [6]. Higher levels of active MAPK have been found to levels were correlated with a lower overall survival (OS) rate
be associated with metastasis to the lymph nodes [7]. Another and that high pMAPK levels were correlated with a higher re-
study showed that TNBCs with overexpression of active lapse-free survival (RFS) rate.
MAPK (phosphorylated [p]MAPK) and low MAPK scores
(which reflect weak immunohistochemical staining and low MATERIALS AND METHODS
tumor grade) were associated with a higher recurrence rate [4].
However, because of the small number of TNBCs in that study, Patient Selection
the role of the MAPK signaling pathway in TNBC was not well This retrospective study involved 97 patients with TNBC and
characterized. Likewise, the expression and prognostic signif- 223 patients with non-TNBC. The solid breast tumors were ob-
icance of the ERK subtypes (isoforms) ERK-1 and ERK-2 in tained from the breast tissue frozen-tumor banks at The Uni-
TNBC have not been well defined. versity of Texas MD Anderson Cancer Center and Hospital
Phospho-enriched protein in astrocytes (PEA)-15 is a Clínico Universitario de Valencia. The use of tissue blocks in
novel 15-kDa protein that blocks the activity of ERK by in- the creation of the RPPAs, use of RPPAs, and chart review for
hibiting the transcription factor Elk-1, which regulates this study were approved by the institutional review boards of
ERK-dependent transcription [8 –11]. PEA-15 binding to MD Anderson Cancer Center and Hospital Clínico Universi-
ERK may be regulated by phosphorylation [9, 10, 12–14]. tario de Valencia.

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Akt has been shown to activate mammalian target of rapa- Our retrospective analysis included only patients who had
mycin (mTOR), which phosphorylates and activates p70S6 invasive ductal or invasive lobular carcinoma and a disease
kinase. Akt also regulates the phosphorylation of PEA-15 at stage of I, II, or III. Patient ages, histological tumor types, tu-
serine 116 (S116) both in vitro and in vivo, stabilizing its mor grades, and disease stages were compared among patient
antiapoptotic function [15]. The phosphorylation of both subgroups defined on the basis of hormone receptor and
S104 and S116 on PEA-15 is required to block the binding HER-2 status.
of PEA-15 to ERK and thereby regulate the MAPK cascade
[16]. Thus, PEA-15’s state of phosphorylation may affect Reverse-Phase Protein Lysate Microarray
its modulation of ERK activity in TNBC cells. Indeed, loss Protein was extracted from human breast cancer samples, and
of PEA-15 expression is a marker of the transition from non- lysates were prepared and arrayed as previously described
invasive (ductal carcinoma in situ) to invasive mammary [20 –21]. Each array slide was then probed with the following
epithelial tumors [17]. Further, PEA-15 gene therapy inhib- dilutions of validated primary rabbit antibodies: anti-PEA-15
ited tumor growth in a xenograft model of TNBC, and when (1:1,000) (Cell Signaling Technology, Danvers, MA), rabbit
PEA-15 was overexpressed in TNBC cells, it inhibited cell anti-pPEA-15 (S116) (1:1,000) (Invitrogen, Carlsbad, CA),
proliferation, sequestered ERK in the cytoplasm, and in- rabbit anti–ERK-2 (1:250) (Cell Signaling Technology), rabbit
duced apoptosis through the death effector pathway [18]. anti-pMAPK (1:1,000) (Cell Signaling Technology), rabbit
Tuberous sclerosis protein 2 (TSC-2) is a tumor suppres- anti–MEK-1 (1:15,000) (Epitomics, Inc., Burlingame, CA),
sor and is directly phosphorylated by ERK, resulting in rabbit anti–TSC-2 (1:500) (Epitomics, Inc.), rabbit anti-p70S6
functional inactivation of TSC-2, impairing its ability to in- kinase (1:500) (Epitomics, Inc.), rabbit anti-p27 (1:500) (Santa
hibit mTOR signaling and cell proliferation [19]. Akt has Cruz Biotechnology, Inc., Santa Cruz, CA), rabbit anti-Akt (1:
been shown to activate mTOR, which phosphorylates and 250) (Cell Signaling Technology), or rabbit anti-Aktp308 (1:
activates p70S6 kinase, which is required for cell growth 250) (Cell Signaling Technology), and the signal was
and G1 cell cycle progression. ERK-1 and ERK-2 signaling amplified with a DakoCytomation (Dako, Carpinteria, CA)
can also trigger downregulation of p27 KIP1, a cyclin-depen- catalyzed system.
dent kinase inhibitor that is required for S-phase entry and
cell cycle progression [20]. Statistical Analysis
The objective of the present study was to establish, in more The statistical analysis was based on all 320 patients in the
detail and in a larger population, the clinical significance of study. Descriptive statistics were used to summarize the pa-
ERK (by assessing ERK-2 and pMAPK), its direct targets tients’ demographic and clinical characteristics. The mean,
(MAPK/ERK kinase [MEK]-1, PEA-15, pPEA-15, TSC-2, standard deviation, median, and range were summarized for
p70S6 kinase, and p27), and other related important targets continuous variables, and either the two-sample t-test or the
(Akt and Aktp308) in patients with TNBC versus non-TNBC analysis of variance method was used to compare the continu-
patients. Further, we attempted to address the significance of ous variables among two or more groups. The number of pa-
PEA-15 and its relationship with ERK in TNBC. We measured tients in each level and the corresponding frequencies were
the expression of ERK and its direct targets in patients with provided for categorical variables. The Spearman correlation
TNBC and non-TNBC by reverse-phase protein array (RPPA), coefficient was used to assess relationships between two bio-
a method that allows measurement of a protein in hundreds of markers. We used the Kaplan–Meier method to estimate the
samples using a robust quantification of protein concentrations median OS and RFS times. For the analysis of OS times, death
over a dynamic range. We correlated the relative expression of was counted as an event, and patients who survived were
ERK and its direct targets with the pathological and clinical counted as censored at their date of last follow-up. The OS time
variables of patients with TNBC and found that high ERK-2 was calculated as the interval between the diagnosis date and

www.TheOncologist.com
768 Role of ERK in Triple-Negative Breast Cancer

Table 1. Clinical characteristics of breast cancer patients according to HR status

Triple-negative, HR"HER-2#, HR#HER-2", HR"HER-2",
Characteristic n (%) n (%) n (%) n (%) p-valuea
Age at diagnosis, yrs
!50 37 (38.1) 26 (12.6) 0 (0) 1 (10.0) !.0001
"50 60 (61.9) 180 (87.4) 7 (100) 9 (90.0)
Histopathological diagnosis
Invasive ductal carcinoma 94 (96.9) 188 (91.3) 6 (85.7) 9 (90.0) .13
Invasive lobular carcinoma 3 (3.1) 18 (8.7) 1 (14.3) 1 (10.0)
Disease stage
I 20 (20.6) 52 (25.2) 3 (42.9) 3 (30.0) .17
II 58 (59.8) 129 (62.6) 2 (28.6) 7 (70.0)
III 19 (19.6) 25 (12.1) 2 (28.6) 0 (0)

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Nuclear gradeb
1 3 (3.7%) 42 (30.9) 0 (0) 2 (25.0) !.0001
2 10 (12.2%) 76 (55.9) 3 (50.0) 3 (37.5)
3 69 (84.2%) 18 (13.2) 3 (50.0) 3 (37.5)
a
p-values from Fisher’s exact test.
b
Nuclear grade data were not available for all patients.
Abbreviations: HER-2, human epidermal growth factor receptor 2; HR, hormone receptor.

Table 2. Biomarkers in breast cancer patients according to HR status

p-valuea

(a) (a) (a) (b) (b) (c)

Triple-negative (a), HR"HER-2# (b), HR#HER-2" (c), HR"HER-2" (d), versus versus versus versus versus versus
Biomarker median (range) median (range) median (range) median (range) (b) (c) (d) (c) (d) (d)
pMAPK 0.390 (#2.314 to 3.598) 0.238 (#2.640 to 2.998) #0.827 (#1.728 to 0.477) #0.493 (#1.182 to 2.053) .93 .01 .31 .03 .43 0.12

ERK-2 #0.004 (#1.522 to 1.078) 0.192 (#1.935 to 1.146) 0.069 (#0.644 to 0.253) #0.106 (#0.809 to 0.403) .002 .90 .25 .16 .01 .28
MEK-1 0.716 (#4.424 to 3.057) #0.756 (#4.688 to 1.849) 0.023 (#2.748 to 0.912) #0.111 (#3.658 to 1.128) !.0001 .19 .06 .17 .42 .63

PEA-15 #0.070 (#1.112 to 1.277) 0.155 (#0.706 to 1.184) 0.307 (#0.309 to 1.148) 0.101 (#0.550 to 1.328) !.0001 .24 .57 .82 .57 .44
pPEA-15 #0.121 (#2.183 to 3.998) 0.033 (#1.121 to 4.240) #0.215 (#0.600 to 4.039) 1.707 (#0.769 to 4.273) .005 .59 .005 .99 .07 .49

TSC-2 0.076 (#1.951 to 1.288) #0.025 (#2.407 to 1.450) 0.605 (#0.792 to 1.107) 0.081 (#1.070 to 1.016) .15 .06 .84 .03 .43 .20
p27 #0.209 (#0.971 to 1.871) 0.075 (#2.195 to 1.675) #0.076 (#0.633 to 1.524) 0.125 (#0.512 to 2.224) !.0001 .91 .09 .68 .92 .49

Akt 0.096 (#2.571 to 2.013) #0.512 (#3.334 to 2.442) 0.246 (#1.178 to 2.464) #0.115 (#1.975 to 0.589) .002 .45 .23 .10 1.00 .20
Aktp308 #0.116 (#1.697 to 3.331) #0.409 (#2.159 to 1.899) #0.077 (#0.993 to 0.915) #0.505 (#1.022 to 0.434) .0002 .61 .10 .55 .69 .49

a
p-value from Kruskal-Wallis test.
Abbreviations: ERK-2, extracellular signal–related kinase 2; HER-2, human epidermal growth factor receptor 2; HR,
hormone receptor; MEK-1, MAPK/ERK kinase 1; PEA-15, phospho-enriched protein in astrocytes; pPEA-15,
phosphorylated PEA-15; pMAPK, phosphorylated mitogen-activated protein kinase; TSC-2, tuberous sclerosis protein 2.

either the date of death or date of last follow-up for those who estimated the hazard ratio (HR) and 95% confidence interval
survived. For the analysis of RFS times, disease relapse and (CI) for each potential prognostic factor. All prognostic factors
death were considered events. The RFS interval was calculated were then included in a saturated model, and backward elimi-
as the interval between the diagnosis date and the date of dis- nation was used to remove factors from the model on the basis
ease relapse, date of death, or, for patients who did not experi- of the likelihood ratio test in the multiple regression analysis to
ence relapse, date of last follow-up. Cox proportional hazards derive the final multivariate model. The statistical software
regression analysis was then used to examine the relationship programs used in the analysis were SAS 9.2 (SAS Institute
between potential covariates and OS and RFS times. This mod- Inc., Cary, NC) and R version 2.11.1 (R Foundation for Statis-
eling was done in a univariate fashion. From this model, we tical Computing).
Bartholomeusz, Gonzalez-Angulo, Liu et al. 769

Table 3. MAPK pathway-related biomarker correlation in patients with triple-negative breast cancer
Spearman correlation coefficient (p)
Biomarker pPEA-15 ERK-2 pMAPK P70S6 kinase
PEA-15 0.4961 (!.0001) 0.4601 (!.0001) #0.1090 (.29) #0.2319 (.02)
pPEA-15 0.0245 (.81) #0.1675 (.10) #0.4049 (!.0001)
ERK-2 #0.1557 (.13) 0.1340 (.19)
pMAPK #0.0775 (.45)
Abbreviations: ERK-2, extracellular signal–related kinase 2; MAPK, mitogen-activated protein kinase; pMAPK,
phosphorylated mitogen-activated protein kinase; pPEA-15, phosphorylated PEA-15; pMAPK, phosphorylated mitogen-
activated protein kinase.

RESULTS ease stage were significantly associated with the OS time (Ta-

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ble 4), and pMAPK levels were marginally significantly
Clinical and Molecular Characteristics of Patients
associated with the RFS interval (Table 5). Patients with four
with TNBC to nine positive lymph nodes (HR, 6.29; 95% CI, 2.19 –18.04;
We first compared the characteristics of the 97 patients with p % .0006) had a higher risk for death than patients with one to
TNBC with those of the 223 patients with non-TNBC (Table
three or "10 positive nodes, although the latter observation
1). The median ages at diagnosis were 55 years (range, 27– 86)
was based on only seven patients and two deaths.
for patients with TNBC and 67 years (range, 30 – 89) for pa-
On multivariate analysis, patients with stage I or stage II
tients with non-TNBC (Wilcoxon rank-sum test p ! .0001).
disease had a lower risk for death during the study period than
We subdivided patients with non-TNBC into three groups:
patients with stage III disease (Table 6), and patients with high
hormone receptor positive and HER-2#, hormone receptor
ERK-2– expressing tumors had a higher risk for death during
negative and HER-2$, and triple positive. Whereas most
the study period than patients with low ERK-2– expressing tu-
TNBC tumors had a nuclear grade of 3 (84%), most tumors that
mors. On multivariate analysis, high pMAPK levels were as-
were hormone receptor positive and HER-2# had a nuclear
sociated with a significantly higher RFS rate in patients with
grade of 2 (56%). Invasive ductal carcinoma was the most
TNBC (Table 7). Thus, ERK-2 was a significant predictor of
common histological type for both TNBC (97%) and non-
the OS time and pMAPK was a significant predictor of the RFS
TNBC (91%) tumors.
interval in TNBC patients. Figures 1– 4 show the
We observed that, compared with patients with hormone
Kaplan–Meier survival curves for OS and RFS probabilities by
receptor–positive and HER-2# breast cancer, patients with
ERK-2 and pMAPK expression levels for patients with TNBC.
TNBC had significantly lower levels of ERK-2, PEA-15,
The median OS time was 4.08 years among patients with high
pPEA-15, and p27 and higher levels of MEK-1, Akt, and
ERK-2 levels and was not available for patients with low
Aktp308. The pMAPK level was higher in the TNBC and
ERK-2 levels (p % .05) (Fig. 1). The 5-year OS rates were 57%
hormone receptor–positive and HER-2# groups than in the
among patients with high ERK-2 levels and 79% among pa-
hormone receptor–negative and HER-2$ group. In addition,
tients with low ERK-2 levels. For the direct targets of ERK
the mean expression level of PEA-15 was lower in patients
(MEK-1, PEA-15, pPEA-15, TSC-2, p70S6 kinase, and p27),
with TNBC than in patients with triple-positive breast can-
the differences in OS and RFS outcomes between patients with
cer (Table 2).
high expression and patients with low expression were not sig-
To assess the relationship between ERK and PEA-15,
nificant in patients with TNBC.
pPEA-15, and p70S6 kinase in patients with TNBC, Spearman
In patients with non-TNBC, the median OS time was 16.42
correlation coefficients were calculated. We observed positive
years, and the median RFS interval was 14.58 years. Age at
correlations between PEA-15 and ERK-2 (p ! .0001) and be-
diagnosis, disease stage, and normalized p70S6 kinase values
tween PEA-15 and pPEA-15 (S116) (p ! .0001) (Table 3) and
were significantly associated with the OS time in patients with
negative correlations between PEA-15 and p70S6 kinase (p %
non-TNBC. Patients with stage I disease had a lower risk for
0.02) and between pPEA-15 and p70S6 kinase (p ! .0001).
death during the study period than patients with stage III dis-
ease, and patients with higher normalized p70S6 kinase values
Outcomes in TNBC and non-TNBC Patients
had a higher risk for death than those with lower normalized
In the patients with TNBC, the median follow-up time was
p70S6 kinase values. Patients aged "50 years also had a higher
2.47 years (range, 0.25–19.45 years). Twenty-four of the 97
risk for death than patients aged !50 years.
patients had died by the time of the analysis. The median OS
time was 7.08 years. Thirty-one of the 97 patients with TNBC
had relapsed by the time of the analysis. The median RFS in- DISCUSSION
terval was 5.38 years (range, 0.25–19.45 years). Our results demonstrate that high ERK (ERK-2 and pMAPK)
On univariate analysis, ERK-2 expression level and dis- expression is an important predictor of OS and disease-free

www.TheOncologist.com
770 Role of ERK in Triple-Negative Breast Cancer

Table 4. Univariate Cox proportional hazards model for overall survival time in patients with triple-negative
breast cancer
n of patients Median survival, Hazard ratio
Prognostic factor (deaths) yrs p-value (95% CI)
Age at diagnosis, yrs
!50 37 (6) 1.00
"50 60 (18) 7.08 .48 1.40 (0.55–3.59)
n of affected nodes
0 52 (8)
1–3 26 (6) 5.44 .18 2.09 (0.70–6.23)
4–9 12 (8) 3.00 .0006 6.29 (2.19–18.04)
"10 7 (2) 4.92 .23 2.66 (0.55–12.97)
Histopathologic diagnosis

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Invasive ductal carcinoma 94 (24) 7.08 1.0
Invasive lobular carcinoma 3 (0) .99
Disease stage
I 20 (1) .003 0.04 (0.005–0.33)
II 58 (14) 6.17 .006 0.29 (0.12–0.70)
III 19 (9) 2.52 1.00
Nuclear gradea
1 3 (1) .74 0.71 (0.09–5.55)
2 10 (2) .22 0.38 (0.08–1.77)
3 69 (13) 1.00
Molecular marker expression
ERK-2 97 (24) 7.08 .03 3.5 (1.17–10.47)
!0.110471 58 (9) 1.00
"0.110471 39 (15) 4.08 .06 2.21 (0.96–5.05)
pMAPK 97 (24) 7.08 .09 0.69 (0.45–1.06)
MEK-1 97 (24) 7.08 .53 0.94 (0.77–1.15)
PEA-15 97 (24) 7.08 .27 0.54 (0.18–1.62)
pPEA-15 97 (24) 7.08 .15 0.79 (0.57–1.09)
TSC-2 97 (24) 7.08 .99 0.996 (0.59–1.68)
p27 97 (24) 7.08 .6 0.82 (0.4–1.70)
a
Nuclear grade data were not available for all patients.
Abbreviations: CI, confidence interval; ERK-2, extracellular signal–related kinase 2; MEK-1, MAPK/ERK kinase 1; PEA-
15, phospho-enriched protein in astrocytes; pPEA-15, phosphorylated PEA-15; pMAPK, phosphorylated mitogen-activated
protein kinase; TSC-2, tuberous sclerosis protein 2.

survival outcomes in patients with TNBC and suggest that activity was associated with a shorter disease-free survival in-
ERK expression may be the driving force in TNBC, in contrast terval in patients with breast cancer [7, 22–24]. Oncogenic ac-
to non-TNBC, in which the driving force is HER-2 or estrogen tivation of ERK resulting in persistent activation of the ERK–
receptor expression. Our comparison of the mean levels of ex- MAPK cascade occurs primarily through mutationally
pression of ERK and its direct targets in TNBC and non-TNBC activated Ras and B-Raf and/or epidermal growth factor recep-
tumors also confirmed the importance of the ERK pathway in tor (EGFR) and HER-2 overexpression [25, 26]. The chemo-
TNBC. therapeutic agent paclitaxel activates the antiapoptotic MEK–
ERK-1 and ERK-2 are dually phosphorylated by MEK on ERK signaling pathway, and thus MEK and ERK levels or
threonine and tyrosine residues. ERK is a downstream target activation could affect how well paclitaxel induces apoptosis
for receptor tyrosine kinases and for Ras, which is mutated in [27, 28]. MAPK overexpression was previously shown to be
many cancers. The oncogenic potential of ERK was demon- associated with both a high recurrence rate in patients with
strated in part by the finding that elevated ERK-1 or ERK-2 TNBC and a low survival rate following relapse [4].
Bartholomeusz, Gonzalez-Angulo, Liu et al. 771

Table 5. Univariate Cox proportional hazards model for relapse-free survival time in patients with triple-negative
breast cancer
n of patients Hazard ratio
Prognostic factor (deaths/relapses) Median survival, yrs p-value (95% CI)
Age at diagnosis, yrs
!50 37 (11) 5.38 1.0
"50 60 (20) .98 0.99 (0.47–2.08)
n of affected nodes
0 52 (10) – – –
1–3 26 (7) – .26 1.74 (0.66–4.59)
4–9 12 (9) 1.36 .0001 6.06 (2.44–15.05)
"10 7 (5) 2.40 .005 4.63 (1.58–13.58)
Histopathologic diagnosis

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Invasive ductal carcinoma 94 (28) 1.0
Invasive lobular carcinoma 3 (3) 1.55 .09 2.82 (0.85–9.33)
Disease stage
I 20 (1) .0007 0.03 (0.004–0.22)
II 58 (16) !.0001 0.22 (0.10–0.45)
III 19 (14) 1.55 1.00
Nuclear gradea
1 3 (1) 0.51 0.51 (0.07–3.86)
2 10 (3) 0.23 0.47 (0.13–1.62)
3 69 (20) 1.00
Molecular marker expression 97
ERK-2 97
!0.110471 58 (17) 5.38 1.00
"0.110471 39 (14) 0.54 1.25 (0.62–2.53)
pMAPK 97 (31) 5.38 0.08 0.72 (0.51–1.04)
MEK-1 97 (31) 5.38 0.38 1.10 (0.90–1.34)
PEA-15 97 (31) 5.38 0.88 0.93 (0.37–2.34)
pPEA-15 97 (31) 5.38 0.17 0.81 (0.60–1.10)
TSC-2 97 (31) 5.38 0.59 1.14 (0.71–1.80)
p27 97 (31) 5.38 0.37 0.73 (0.36–1.46)
a
Nuclear grade data were not available for all patients.
Abbreviations: CI, confidence interval; ERK-2, extracellular signal–related kinase 2; MEK-1, MAPK/ERK kinase 1; PEA-
15, phospho-enriched protein in astrocytes; pPEA-15, phosphorylated PEA-15; pMAPK, phosphorylated mitogen-activated
protein kinase; TSC-2, tuberous sclerosis protein 2.

In our study, patients with TNBC with high ERK-2– ex- ger RFS and OS times, suggesting that the cellular localization
pressing tumors had a shorter OS duration. However, patients of pmTOR may play an important role in patient outcomes
with TNBC with high pMAPK levels had a significantly [29]. Thus, it would be relevant to perform immunohistochem-
higher RFS rate. We speculate that the location of ERK and ical studies to determine the cellular localization of ERK.
pMAPK may have contributed to physiological responses in PEA-15 levels correlated positively with ERK levels, con-
breast cancer cells, affecting patient outcomes. The RPPA ap- sistent with recent findings that PEA-15 leads to prolonged ty-
proach used in our current study did not provide information rosine phosphorylation of fibroblast growth factor receptor
about whether ERK and pMAPK were located in the cyto- substrate 2#, resulting in activation of MEK-1 and MEK-2 and
plasm or the nucleus. A recent study involving patients with ERK-1 and ERK-2 [30]. This is consistent with our study and
gastric cancer showed that cytoplasmic pmTOR expression other studies that have shown that PEA-15 can induce the ex-
was associated with significantly shorter RFS and OS times, pression of ERK and pMAPK [13, 31]. Interestingly, we found
whereas nuclear pmTOR expression was associated with lon- a negative correlation between PEA-15 and p70S6 kinase and

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772 Role of ERK in Triple-Negative Breast Cancer

Table 6. Multivariate Cox proportional hazards model for

overall survival time in triple-negative breast cancer
patients (n % 97)
Variable Hazard ratio (95% CI) p-value
Disease stage
I 0.03 (0.004–0.26) .002
II 0.28 (0.12–0.68) .005
III 1.00
ERK-2 expression
!0.110471 1.00
"0.110471 2.76 (1.19–6.41) .02
Abbreviations: CI, confidence interval; ERK-2, Figure 2. Relapse-free survival probability by extracellular sig-
extracellular signal–related kinase 2. nal–related kinase 2 status in patients with triple-negative breast

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cancer.
Abbreviation: NA, not available.

Table 7. Multivariate Cox proportional hazards model for

relapse-free survival time in triple-negative breast cancer
patients (n % 97)
Variable Hazard ratio (95% CI) p-value
Disease stage
I 0.03 (0.003–0.19) .0004
II 0.20 (0.10–0.42) !.0001
III 1.00
pMAPK expression 0.66 (0.46–0.95) .03
Abbreviations: CI, confidence interval; pMAPK,
phosphorylated mitogen-activated protein kinase.

Figure 3. Overall survival probability by phosphorylated mito-

gen-activated protein kinase status in patients with triple-negative
breast cancer.

Figure 1. Overall survival probability by extracellular signal–

related kinase 2 status in patients with triple-negative breast
cancer.
Abbreviation: NA, not available. Figure 4. Relapse-free survival probability by phosphorylated
mitogen-activated protein kinase status in patients with triple-
negative breast cancer.
between pPEA-15 (S116) and p70S6 kinase. Overexpression
of p70S6 kinase is known to be associated with aggressive dis-
ease and a poor prognosis in patients with breast cancer. We press low levels of p70S6kinase. Further studies are needed to
previously showed that high PEA-15 expression is associated confirm this hypothesis.
with a longer OS time in patients with ovarian cancer. Thus, we PEA-15 is not known to impair the activation or the enzy-
can hypothesize that patients with high PEA-15 expression ex- matic activity of ERK, although it can sequester ERK in the
Bartholomeusz, Gonzalez-Angulo, Liu et al. 773

cytoplasm, thereby preventing ERK’s phosphorylation. tic target in TNBC. It would be relevant to develop MAPK
PEA-15 may act as a master regulator of ERK by regulating pathway signatures in TNBC that can be used for finding novel
ERK’s cellular localization. Furthermore, PEA-15 may con- therapeutic interventions, predicting prognosis, and optimiz-
trol cell proliferation by preventing ERK accumulation in the ing treatment options. We are planning further immunohisto-
nucleus. Although certain cancer and normal cells proliferate chemical studies to determine the cellular localization of ERK
rapidly while expressing PEA-15 at high levels [32, 33], we and pMAPK and are developing a MEK–ERK pathway signa-
previously found that high levels of PEA-15 protein expres- ture in TNBC cells that could be used for patient selection for
sion in women with ovarian cancer were independently asso- treatment with a MEK inhibitor.
ciated with a higher OS rate [31]. In breast cancer patients, low
PEA-15 expression was previously linked with aggressive ACKNOWLEDGMENTS
forms of the disease. Similarly, greater PEA-15 expression We thank Bryan Tutt and Sunita Patterson of the Department
was associated with less invasion in breast cancer through ef- of Scientific Publications at The University of Texas MD An-
fects on the ERK pathway [17]. We also found that PEA-15 derson Cancer Center for their expert editorial assistance. We
expression levels were lower in high-grade than in low-grade also are grateful to Dr. Naoto Ueno for critical review of the
breast tumors. manuscript.
The ERK–MAPK pathway could become an important

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This work was supported by National Cancer Institute
therapeutic target in TNBC for several reasons. First, elevated grant 1K99CA139006-01A1 (C.B.) and the Nellie B. Connally
MAPK activity correlates with shorter survival times in breast Breast Cancer Research Fund (Breast Cancer Translational
cancer patients. Second, aberrant activation of the MAPK Research Laboratory).
pathway in human cancer cells occurs by upstream activation This work was presented in part at the American Associa-
by EGFR and Ras small GTPase. Third, ERK promotes cell tion for Cancer Research meeting, Washington, DC, April
proliferation, cell survival, epithelial–mesenchymal transition, 2010.
angiogenesis, and metastasis. Finally, the MEK–ERK signal-
ing pathway has been shown to play a critical role in the sur-
AUTHOR CONTRIBUTIONS
vival and growth of breast cancer cells [34]. Conception/Design: Chandra Bartholomeusz
Provision of study material or patients: Ana M. Gonzalez-Angulo, Ana
CONCLUSION AND SUMMARY Lluch, Jaime Ferrer-Lozano
Collection and/or assembly of data: Ana M. Gonzalez-Angulo, Ana Lluch,
We found, in the current study, that patients with TNBC with Jaime Ferrer-Lozano
high levels of ERK-2 had shorter OS times than patients with Data analysis and interpretation: Chandra Bartholomeusz, Ping Liu, Naoki
Hayashi
TNBC with low levels of ERK-2, and that high pMAPK levels Manuscript writing: Chandra Bartholomeusz, Gabriel N. Hortobágyi
correlated with a higher RFS rate. Our data demonstrate that Final approval of manuscript: Chandra Bartholomeusz, Ana M.
Gonzalez-Angulo, Ana Lluch, Ping Liu, Gabriel N. Hortob ágyi, Jaime
ERK and its signaling pathway may be an important therapeu- Ferrer-Lozano, Naoki Hayashi

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