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MicroRNA 603 acts as a tumor suppressor and inhibits triple-
negative breast cancer tumorigenesis by targeting elongation
factor 2 kinase
Recep Bayraktar
1,2
, Martin Pichler
4
, Pinar Kanlikilicer
1
, Cristina Ivan
1,4
, Emine
Bayraktar
1,2
, Nermin Kahraman
1
, Burcu Aslan
1
, Serpil Oguztuzun
3
, Mustafa
Ulasli
2
, Ahmet Arslan
2
, George Calin
1,4
, Gabriel Lopez-Berestein
1,4
, Bulent
Ozpolat
1,4
1
Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
2
Department of Medical Biology, School of Medicine, Gaziantep University, Gaziantep, Turkey
3
Department of Biology, Kirikkale University, Kirikkale, Turkey
4
Center for RNA Interference and Non-Coding RNAs, The University of Texas MD Anderson Cancer Center, Houston, Texas,
USA
Correspondence to: Bulent Ozpolat, email: Bozpolat@mdanderson.org.
Keywords: eEF2K, triple negative breast cancer, liposomes, nanoparticles, miR-603
Received: June 29, 2016 Accepted: November 30, 2016 Published: December 27, 2016
ABSTRACT
Triple negative breast cancer (TNBC) is an aggressive type of breast cancer
characterized by the absence of dened molecular targets, including estrogen
receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor
2 (HER2) and is associated with high rates of relapse and distant metastasis despite
surgery and adjuvant chemotherapy. The lack of effective targeted therapies for
TNBC represents an unmet therapeutic challenge. Eukaryotic elongation factor 2
kinase (eEF2K) is an atypical calcium/calmodulin-dependent serine/threonine kinase
that promotes TNBC tumorigenesis, progression, and drug resistance, representing
a potential novel molecular target. However, the mechanisms regulating eEF2K
expression are unknown. Here, we report that eEF2K protein expression is highly
up-regulated in TNBC cells and patient tumors and it is associated with poor patient
survival and clinical outcome. We found that loss/reduced expression of miR-603
leads to eEF2K overexpression in TNBC cell lines. Its expression results in inhibition
of eEF2K by directly targeting the 3-UTR and the inhibition of tumor cell growth,
migration and invasion in TNBC. In vivo therapeutic gene delivery of miR-603 into
TNBC xenograft mouse models by systemic administration of miR-603-nanoparticles
led to a signicant inhibition of eEF2K expression and tumor growth, which was
associated with decreased activity of the downstream targets of eEF2K, including Src,
Akt, cyclin D1 and c-myc. Our ndings suggest that miR-603 functions as a tumor
suppressor and loss of miR-603 expression leads to increase in eEF2K expression and
contributes to the growth, invasion, and progression of TNBC. Taken together, our
data suggest that miR-603-based gene therapy is a potential strategy against TNBC.
INTRODUCTION
Breast cancer (BC) is the most common malignancy
among women and the second leading cause of cancer-
related deaths worldwide [1]. Every year in the United
States, nearly 232,000 cases of invasive breast cancer and
65,000 cases of in situ breast cancer are diagnosed, and
more than 40,000 women die to breast cancer [2]. BC is
a highly complex and heterogeneous disease with distinct
biological and clinical behaviors [3]. BC is classied into
ve major subtypes according to molecular features and
intrinsic expression of the genes encoding the estrogen
www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 7), pp: 11641-11658
Research Paper
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receptor (ER), progesterone receptor (PR), and human
epidermal growth factor receptor 2 (HER2): luminal A (ER
and/or PR positive and HER2 negative), luminal B (ER or
PR positive and HER2 positive), HER2 overexpressing,
normal-breast like and basal-like or triple-negative breast
cancer (TNBC) phenotype [3].
TNBC accounts for approximately 10-20% of all
cases of breast cancer and is characterized by the absence
of yet dened molecular targets, including estrogen
receptor (ER), progesterone receptor (PR) and human
epidermal growth factor receptor 2 (HER2) [4, 5]. Thus,
therapies targeting ER (i.e., tamoxifen) and HER2 (also
known as eERB2) (i.e., trastuzumab) are ineffective
against TNBC [5]. The other important characteristics
of TNBC include aggressive clinical behavior, early
relapses, and metastasis as well as reduced sensitivity to
conventional therapies. The poor clinical outcome and
short overall patient survival predominantly attributed
to intratumoral heterogeneity and mutated TP53, which
is detected in up to 84% of TNBC cases [5–7]. A better
understanding of the biology of TNBC and the underlying
molecular mechanisms are needed to identify novel
therapeutic targets and develop highly effective targeted
therapies for improved patient outcomes [8–10].
Recently, emerging evidence has revealed that
eukaryotic elongation factor 2 kinase (eEF2K) is a
potential molecular driver in several cancers, including
pancreatic, brain and breast cancer [11–16]. eEF2K is
the only calcium/calmodulin activated member of the
α-kinase family and is considered an atypical kinase since
its catalytic domain is not structurally similar to those of
conventional protein kinases [17, 18]. eEF2K activity is
regulated by multiple mechanisms to control the rate of
protein chain elongation by phosphorylating/inactivating
eEF2 (at threonine 56), which mediates the movement
of the ribosome on transfer RNA (tRNA) from the A site
to the P site [19–23]. eEF2K promotes cell survival in
conditions of nutrient deprivation, hypoxia and metabolic
stress by regulating the rate of translation [24]. Recently,
eEF2K was shown to promote cell proliferation, cell
migration, invasion, epithelial-mesenchymal transition
(EMT) and TNBC tumorigenesis and progression through
modulating the cell cycle (G1/S transition) by regulating
cyclin D1, c-myc, PI3K/Akt, Src/Fak and insulin-like
growth factor receptor (IGFR) signaling [11, 13, 14,
16]. Therapeutic targeting of eEF2K triggers apoptosis
and suppresses TNBC tumor growth, in addition to
and increased doxorubicin and paclitaxel efcacy [16].
These reports suggest that eEF2K is a critical factor
for breast cancer progression and the strategies aimed
at manipulating the activity of eEF2K may aid the
development of novel treatment regimens for TNBC.
Non-coding RNAs such as microRNAs (miRNA)
have emerged as new regulators of gene expression
across various biological processes, including cell cycle
regulation, differentiation, metabolism and aging. miRNAs
are involved in many diseases such as cardiovascular
and neurodegenerative diseases and but they also play
an a vital role in the pathogenesis of human cancers and
clinical applications as therapeutics [25]. miRNAs can
be used to classify a wide variety of human cancers with
particular signatures and dene the molecular architecture
of human cancers [26–30]. miRNAs are 18-25 nucleotides
in length and regulate the expression of target genes at
the posttranscriptional level through interaction with
complementary sequences usually found in the 3’-UTRs
of target mRNAs, resulting in inhibition of translation and/
or in mRNA degradation [25].
Several oncogenic and tumor suppressor miRNAs
have been identied as the promoters of tumor formation
and growth when aberrantly expressed in various cancers
[27]. Oncogenic miRs are frequently over-expressed
in cancer tissues, including miR-21, miR-17-92, miR-
155 and miR-372, while miRs such as miR-34 and the
let-7 family miR-15a and miR-16-1 are considered as
tumor suppressors and their expression is often reduced
in cancer tissues [27]. Over the years of studies, growing
evidences have shown that miRNAs have an important
role in breast cancer progression, invasion, angiogenesis
and metastasis. Apparently, some miRNAs functionally
take part in several important cell proliferation pathways
such Src, and MAPK and the aberrant expression of these
miRNAs is responsible for evading growth suppressors
and sustaining proliferative signaling in breast cancer cells
[31–33]. miRNA 603 (miR-603) was recently identied
through genome-wide analysis in glioblastoma [34] and
thyroid cancer with a potential role in cell transformation
[35]. However the role of miR-603 in breast cancer has
not been characterized previously and was the focus of
the current study.
This study demonstrates for the rst time that
miR-603 functions as tumor suppressor in TNBC and
its expression plays an important role in TNBC cell
proliferation, migration/invasion, and tumorigenesis
through the regulation of eEF2K. Our data also suggest
that miR-603-based gene therapy or eEF2K targeted
approaches may be a potential therapeutic strategy against
TNBC.
RESULTS
eEF2K expression is upregulated in TNBC cell
lines and patient tumors
To evaluate eEF2K expression in TNBC we
determined the protein expression level of eEF2K in
TNBC cell lines and patient tumors by Western blot and
immunohistochemistry, respectively. The expression levels
of eEF2K protein were signicantly higher in MDA-
MB-436, MDA-MB-468, MDA-MB-231, BT-20, and
BT-549 cells than in the non-tumorigenic normal breast
cell epithelium MCF-10A cells (Figure 1A). We also
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Figure 1. eEF2K protein and mRNA is overexpressed in TNBC cell lines and patients tumor samples. A. eEF2K protein
expression levels in TNBC cell lines were higher than in the normal breast epithelial cells (MCF-10A). B. The expression levels of eEF2K
in patient tumor tissues and adjacent normal tissues were determined by immunohistochemistry C. High protein expression of eEF2K was
associated with poor overall survival in breast cancers patients with low and high eEF2K expression, log-rank p = 0.0398) as determined
by Kaplan-Meier analysis. D. Relative expression levels of eEF2K mRNA in TNBC cell lines were analyzed with qPCR. E. The algorithms
including TargetScan, miRDB, Diana microT, and microRNA predict that the 3’-UTR of eEF2K is targeted by miR-603. F. Relative
expression levels of miR-603 in TNBC cell lines and in MCF-10A cells were quantied by qPCR using specic primers. The data were
normalized to the expression of U6 small nuclear RNA and are shown as means with SDs for three independent experiments.
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investigated eEF2K expression in 9 TNBC patient and 10
normal breast tissue biopsy samples. The expression levels
of eEF2K protein were highly positive in TNBC patient
biopsy samples (66.66% 6 and 9 patient tumors) than
in normal breast tissues (Figure 1B and Supplementary
Figure 1A).
eEF2K expression is associated with poor
prognosis and breast cancer patient survival
To elucidate the clinical signicance of eEF2K
protein expression, we rst analyzed TCGA database and
determined the prognostic value of eEF2K in 58 breast
cancer patients with basal subtype. Overall survival curves
were plotted according to eEF2K gene expression levels
using the Kaplan-Meier method. Overall survival rate was
signicantly lower in patients with high eEF2K expression
than in patients with low eEF2K expression (p = 0.0398)
(Figure 1C).
miR-603 expression in TNBC is inversely
correlated with eEF2K mRNA expression
We rst analyzed the levels of eEF2K mRNA
in TNBC cell lines, including MDA-MB-231, MDA-
MB-436, BT-20, BT-549 and MDA-MB-468 cell lines
and the non-tumorigenic cell line MCF-10A via qPCR.
The results showed that eEF2K mRNA levels were
upregulated in TNBC cell lines (Figure 1D). To identify
miRNAs that target and regulate eEF2K gene expression,
we used four different algorithms that predict the mRNA
targets of miRNAs: TargetScan (http://www.targetscan.
org/cgi-bin/targetscan/vert_70), miRDB (http://mirdb.org/
cgi-bin/search.cgi), microRNA.org (http://www.microrna.
org/microrna/searchGenes.do), and Diana microT (http://
diana.imis.athena-innovation.gr/DianaTools/) to select
miRNAs that may target eEF2K. Based on this target
prediction strategy, miR-603 was the common miRNA that
targets eEF2K in all four database (Figure 1E). We also
selected miR-3613-3p and miR-3163 that were common in
the three of the databases for binding scores for targeting
eEF2K (Figure 1E).
The basal expression levels of miR-603, miR-3613-
3p, and miR-3163 in TNBC cell lines (MDA-MB-231,
MDA-MB-436, and BT-20) were compared to their
expression levels in MCF-10A cells by qPCR. Only miR-
603 expression was lower in all three TNBC cell lines
than in MCF-10A cells (Figure 1F), suggesting an inverse
relationship of miR-603 to eEF2K and potential role for
miR-603 directly binding eEF2K mRNA and regulating its
expression. The basal expression levels of miR-3613-3p
and miR-3163 did not show any correlation with eEF2K
expression in TNBC cell lines (Supplementary Figure
1B and 1C). Therefore, we hypothesized based on target
prediction tools and the inverse correlation that eEF2K is
a direct target of miR-603 in human TNBC cells.
miR-603 expression suppresses eEF2K in TNBC
cells
Since miR-603 is markedly downregulated in
TNBC cell lines, we ectopically overexpressed miR-
603 in MDA-MB-231, MDA-MB-436, and BT-20 cells
and measured changes in the expression of eEF2K
mRNA and protein levels. As shown in Supplementary
Figure 2A, in miR-603 mimic-transfected MDA-
MB-231, MDA-MB-436, and BT-20 cells, miR-603
was expressed at levels that were 18, 5.1, and 14 times
higher, respectively, than in the corresponding cell
lines transfected with the control miRNA. Ectopic
overexpression of miR-603 signicantly suppressed
the eEF2K mRNA levels in MDA-MB-231, MDA-
MB-436 cells and BT-20 cells (Figure 2A). We further
demonstrated by western blot that miR-603 expression
also led to reduced eEF2K protein expression in MDA-
MB-231 (59.8% reduction), MDA-MB-436 (47.6%
reduction) and BT-20 cells (46.8% reduction) (Figure
2B), indicating that miR-603 is a potential regulator of
eEF2K expression in TNBC cells.
miR-603 regulates eEF2K mRNA expression by
directly binding to 3’-UTR region
To determine whether the negative regulatory
effect of miR-603 on eEF2K expression is mediated
through direct binding to the predicted sites in the 3’-
UTR of the eEF2K mRNA according to four different
algorithms, we evaluated three different predicted miR-
603 binding sites were found in3’-UTR region of eEF2K
gene (Figure 2C). The resulting plasmids were transfected
into HEK293 cells along with the miR-603 mimic or the
scrambled negative-control miRNA. As shown in Figure
2D, luciferase activity was signicantly reduced in cells
transfected with the plasmid containing the binding site 3
in the 3’-UTR of miR-603 (p = 0.03). To further provide
proof of the binding of miR-603 to the specic binding
motifs, we also introduced point mutations (CACTGCC-
>TATGACT) into the corresponding miR-603 binding site
in the 3’-UTR of eEF2K. pEZX-MT06 miRNA reporter
vector containing one point mutation was also used. Only
the mutation in binding site 3 (2037–2065 bp; Figure
2C) completely reversed the effect of overexpression of
miR-603 (Supplementary Figure 2B). We also performed
miRNA luciferase reporter assay in MDA-MB-436
and MDA-MB-231 cells resulting in luciferase activity
was signicantly reduced in cells transfected with the
plasmid containing the binding site 3 in the 3’-UTR of
miR-603 (MDA-MB-436; p < 0.005 and MDA-MB-231;
p<0.05). Collectively, our ndings suggest that binding
site 3 contributes to the interaction between miR-603 and
eEF2K mRNA and that miR-603 directly recognizes and
binds to the eEF2K 3’-UTR to regulate the expression of
the eEF2K mRNA transcript.
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Figure 2. miR-603 negatively regulates eEF2K expression levels in TNBC cells by directly binding to the eEF2K 3’-
UTR. A. miR-603 expression leads to decreased eEF2K mRNA expression levels in MDA-MB-231, MDA-MB-436 and BT-20 in TNBC
cells. Cells lines were analyzed for eEF2K mRNA levels by qPCR 48 h after miR-603 transfection. B. miR-603 expression decreases
eEF2K protein expression levels in MDA-MB-231, MDA-MB-436 and BT-20 cells. TNBC cells were transfected with the miR-603 mimic
or miR-control, and eEF2K protein levels were analyzed by Western blotting 72 h after transfection. Β-Actin was used as a loading control.
Band intensities were quantied using densitometric analysis (right panel, NT, not transfected. C. Three predicted binding sites of miR-603
in the 3’-UTR of human wild-type eEF2K and their sequences. Mutations in the seed sequence of the full-length eEF2K 3’-UTR are shown
in red. D, E. Luciferase reporter assay showed that miR-603 directly targets the eEF2K 3’-UTR-luciferase reporter (wild type or mutant
miR-603 binding sides), in HEK-293, MDA-MB-231 and MDA-MB-436 cells incubated with the miR-603 mimic for 48 h before analysis.
The rey luciferase activity of the reporter was normalized to the internal Renilla luciferase activity. The data are presented as means with
SDs for three independent experiments. *p < 0.05.