Targets of miR-200c mediate suppression of cell motility and anoikis resistance.

Abstract

miR-200c and other members of the miR-200 family promote epithelial identity by directly targeting ZEB1 and ZEB2, which repress E-cadherin and other genes involved in polarity. Loss of miR-200c is often observed in carcinoma cells that have undergone epithelial to mesenchymal transition (EMT). Restoration of miR-200c to such cells leads to a reduction in stem cell-like characteristics, reduced migration and invasion, and increased sensitivity to taxanes. Here we investigate the functional role of novel targets of miR-200c in the aggressive behavior of breast and endometrial cancer cells.
Putative target genes of miR-200c identified by microarray profiling were validated as direct targets using dual luciferase reporter assays. Following restoration of miR-200c to triple negative breast cancer and type 2 endometrial cancer cell lines that had undergone EMT, levels of endogenous target mRNA and respective protein products were measured. Migration and sensitivity to anoikis were determined using wound healing assays or cell-death ELISAs and viability assays respectively.
We found that restoration of miR-200c suppresses anoikis resistance, a novel function for this influential miRNA. We identified novel targets of miR-200c, including genes encoding fibronectin 1 (FN1), moesin (MSN), neurotrophic tyrosine receptor kinase type 2 (NTRK2 or TrkB), leptin receptor (LEPR), and Rho GTPase activating protein 19 (ARHGAP19). These targets all encode proteins normally expressed in cells of mesenchymal or neuronal origin; however, in carcinoma cells that lack miR-200c they become aberrantly expressed and contribute to the EMT phenotype and aggressive behavior. We showed that these targets are inhibited upon restoration of miR-200c to aggressive breast and endometrial cancer cells. We demonstrated that inhibition of MSN and/or FN1 is sufficient to mediate the ability of miR-200c to suppress cell migration. Lastly, we showed that targeting of TrkB mediates the ability of miR-200c to restore anoikis sensitivity.
miR-200c maintains the epithelial phenotype not only by targeting ZEB1/2, which usually facilitates restoration of E-cadherin expression, but also by actively repressing a program of mesenchymal and neuronal genes involved in cell motility and anoikis resistance.

Full text

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RESEARCH ARTICLEOpen Access
Targets of miR-200c mediate suppression of cell
motility and anoikis resistance
Erin N Howe1, Dawn R Cochrane2and Jennifer K Richer1*
Abstract
Introduction: miR-200c and other members of the miR-200 family promote epithelial identity by directly targeting
ZEB1 and ZEB2, which repress E-cadherin and other genes involved in polarity. Loss of miR-200c is often observed
in carcinoma cells that have undergone epithelial to mesenchymal transition (EMT). Restoration of miR-200c to
such cells leads to a reduction in stem cell-like characteristics, reduced migration and invasion, and increased
sensitivity to taxanes. Here we investigate the functional role of novel targets of miR-200c in the aggressive
behavior of breast and endometrial cancer cells.
Methods: Putative target genes of miR-200c identified by microarray profiling were validated as direct targets
using dual luciferase reporter assays. Following restoration of miR-200c to triple negative breast cancer and type 2
endometrial cancer cell lines that had undergone EMT, levels of endogenous target mRNA and respective protein
products were measured. Migration and sensitivity to anoikis were determined using wound healing assays or cell-
death ELISAs and viability assays respectively.
Results: We found that restoration of miR-200c suppresses anoikis resistance, a novel function for this influential
miRNA. We identified novel targets of miR-200c, including genes encoding fibronectin 1 (FN1), moesin (MSN),
neurotrophic tyrosine receptor kinase type 2 (NTRK2 or TrkB), leptin receptor (LEPR), and Rho GTPase activating
protein 19 (ARHGAP19). These targets all encode proteins normally expressed in cells of mesenchymal or neuronal
origin; however, in carcinoma cells that lack miR-200c they become aberrantly expressed and contribute to the
EMT phenotype and aggressive behavior. We showed that these targets are inhibited upon restoration of miR-200c
to aggressive breast and endometrial cancer cells. We demonstrated that inhibition of MSN and/or FN1 is sufficient
to mediate the ability of miR-200c to suppress cell migration. Lastly, we showed that targeting of TrkB mediates
the ability of miR-200c to restore anoikis sensitivity.
Conclusions: miR-200c maintains the epithelial phenotype not only by targeting ZEB1/2, which usually facilitates
restoration of E-cadherin expression, but also by actively repressing a program of mesenchymal and neuronal
genes involved in cell motility and anoikis resistance.
Introduction
Epithelial to mesenchymal transition (EMT) occurs dur-
ing development as it is required for formation of the
neural crest and palate, among other processes [1,2]. In
cancer it is a pathological event associated with tumor
progression and is thought to influence certain steps in
the metastatic cascade, thereby contributing to the
metastatic potential of carcinomas. Specifically, EMT
likely contributes to the ability of carcinoma cells to
invade through basement membrane and stroma and to
intravasate into blood and lymph vessels [3-5]. The pro-
cess of EMT is regulated by several transcription factors,
including Twist, SNAIL, SLUG, ZEB1 (zinc finger E-box
binding homeobox 1) and the closely related SIP1
(ZEB2), as reviewed in [6], which are transcriptional
repressors of E-cadherin.
The miR-200 family of miRNAs, which includes miR-
200c and miR-141 on chromosome 12 and miR-200a/b
and miR-429 on chromosome 1, directly targets ZEB1
and ZEB2 [7-10]. Restoring miR-200c to aggressive
breast, endometrial and ovarian cancer cells substantially
* Correspondence: jennifer.richer@ucdenver.edu
1Program in Cancer Biology, Department of Pathology, University of
Colorado, Anschutz Medical Campus, Mail Stop 8104, P.O. Box 6511, Aurora,
CO, USA
Full list of author information is available at the end of the article
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© 2011 Howe et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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decreases migration and invasion [9-13]. Since ZEB1
represses E-cadherin [14] and other genes involved in
polarity [15], the reduction in migratory and invasive
capacity observed when miR-200c is restored to cancer
cells is widely thought to be due to the ability of miR-
200c to target and repress ZEB1/2 which, in most cases,
allows E-cadherin to be re-expressed. However, even in
cell lines in which E-cadherin is not restored, miR-200c
still dramatically reduces migration and invasion [11],
implying that additional miR-200c targets can facilitate
its ability to suppress cell motility.
We identify and confirm novel direct targets of miR-
200c, including the genes encoding fibronectin 1 (FN1),
moesin (MSN), neurotrophic tyrosine receptor kinase
type 2 (NTRK2 or TrkB), leptin receptor (LEPR), and
Rho GTPase activating protein 19 (ARHGAP19). These
targets are all genes usually expressed in cells of
mesenchymal or neuronal origin. However, in carcinoma
cells that lack miR-200c, repression of these genes is
compromised and they are allowed to be translated and
contribute to an EMT phenotype and aggressive beha-
vior. Here we show that MSN and FN1 are direct targets
of miR-200c that contribute to the ability of miR-200c to
suppress migration. We also identify a completely novel
role for miR-200c - the ability to reverse anoikis resis-
tance and we further pinpoint TrkB as the direct target
that mediates this effect. Anoikis resistance is an impor-
tant, yet understudied, step in the metastatic cascade.
Materials and methods
Cell culture
Hec50 cells were cultured in DMEM with 10% fetal
bovine serum (FBS) and 2 mM L-glutamine. AN3CA
cells and Ishikawa cells were grown in MEM with 5%
FBS, nonessential amino acids (NEAA), penicillin, strep-
tomycin and 1 nM insulin. MCF-7 cells were grown in
DMEM with 10% FBS, and 2 mM L-glutamine. MDA-
MB-231 cells were grown in MEM with 5% FBS,
HEPES, NEAA, 2 mM L-glutamine, penicillin, strepto-
mycin, and insulin. BT549 cells were grown in RPMI
supplemented with 10% FBS and insulin. All cells were
grown in a 37°C incubator with 5% CO2. Cell line iden-
tities were authenticated by isolating genomic DNA
using ZR genomic DNAII kit (Zymo Research, Irvine,
CA, USA) and DNA profiling multiplex PCR was per-
formed using the Identifiler Kit (Applied Biosystems,
Carlsbad, CA, USA) in the UC Cancer Center DNA
Sequencing and Analysis Core.
Transfection
miR-200c (miRNA mimic) or scrambled negative con-
trol (Ambion, Austin, TX, USA) at a concentration of
50 nM were incubated with Lipofectamine 2000 (Invi-
trogen, Carlsbad, CA, USA) in culture medium per the
manufacturer’s instructions before addition to cells.
Cells were incubated at 37°C for 24 hrs before replace-
ment of medium.
DNA and shRNA constructs
pEGP-MSN (created by Stephen Shaw, National Insti-
tutes of Health, purchased from Addgene plasmid
20671, Cambridge, MA, USA) [16]. FN1 was subcloned
from pCR-XL-TOPO-FN1 (Open Biosystems, Catalog
number MHS4426-99240322, Huntsville, AL, USA) into
pcDNA3.1 (Invitrogen). TrkB was subcloned from
pBabe-TrkB (a gift from D. Peeper) into pcDNA3.1.
Microarray analysis
Expression profiling was performed on Hec50 cells
transfected as described above and statistical analysis
was performed as described previously [12]. Array data
have been provided to GEO, accession GSE25332. The
heatmap was generated using GeneSpring GX 11 (Agi-
lent, Santa Clara, CA, USA) and shows genes that are
statistically significantly down-regulated by at least 1.5-
fold in the miR-200c treated samples as compared to
either the mock or scrambled control or both, and are
predicted to be direct targets of miR-200c. Target site
predictions were taken from TargetScan [17], http://
microRNA.org[18], PicTar [19] and MicroCosm [20].
Luciferase assays
A section of the 3’ untranslated region (UTR) of each
target containing the putative binding site(s) for miR-
200c was amplified by PCR from HeLa genomic DNA
using the primers listed in Table S1 in Additional file 1.
Fragments were cloned into the 3’ UTR of a firefly luci-
ferase reporter vector (pMIR-REPORT, Ambion) using
HindIII and SpeI. Mutations in the miR-200c binding
sites were generated by PCR directed mutagenesis.
Mutation primers are listed in Table S1 in Additional
file 1 and introduced mutations are in bold and shown
above the mRNA in each figure. 3’ UTR sequences and
mutations were verified by sequencing. Hec50 cells
(15,000 per well) plated in a 96-well plate were mock
transfected, transfected with negative control, 50 nM
miR-200c, 50 nM miR-200c antagomiR (Dharmacon,
Lafayette, CO, USA)) alone (a200c) or in conjunction
with miR-200c (a200c + 200c). After 24 hrs, the firefly
reporter plasmid (196 ng) and a Renilla luciferase nor-
malization plasmid pRL-SV40 (4 ng) were introduced
using Lipofectamine 2000. Cells were harvested 48 hrs
later for analysis using the Dual Luciferase Reporter
assay system (Promega, Madison, WI, USA)).
Real-time reverse transcription-PCR
RNA was harvested from cells using Trizol (Invitrogen)
and treated with DNase 1 (Invitrogen) for 15 minutes at
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room temperature. RNA was reverse transcribed into
cDNA in a reaction containing reaction buffer, 10 mM
DTT, 1 mM dNTPs, RNase inhibitor (Applied Biosys-
tems), 250 ng random hexamers, and 200 units of
MuLV-RT (Applied Biosystems). For normalization,
real-time reverse transcription-PCR (RT-PCR) was per-
formed on the cDNA using eukaryotic 18S rRNA endo-
genous control primers and FAM-MGB probe (Applied
Biosystems). TaqMan MicroRNA Reverse Transcription
kit was used to generate cDNA for real-time RT-PCR
reaction in conjunction with a miR-200c specific primer
and probe (Applied Biosystems, assay ID 002300). The
reverse transcription primer for miR-200c is a hairpin
primer specific to the mature miRNA and will not bind
to the precursor molecules. For validation of the micro-
array data, SYBR Green real-time RT-PCR was per-
formed using primers specific for each target (primers
listed in Table S1 in Additional file 1). To avoid the
possibility of amplification artifacts, PCR products for all
SYBR Green primer pairs were verified to produce sin-
gle products by agarose electrophoresis and high resolu-
tion melt curve. The relative mRNA or miRNA levels
were calculated using the comparative Ct method
(ΔΔCt). Briefly, the Ct (cycle threshold) values for the
rRNA or actin were subtracted from Ct values of the
target gene to achieve the ΔCt value. The 2−ΔCtwas cal-
culated for each sample and then each of the values was
divided by a control sample to achieve the relative
mRNA or miRNA levels (ΔΔCt).
Immunoblot analysis
Whole-cell protein extracts prepared in RIPA lysis buf-
fer, equalized to 50 μg by Bradford protein assay (Bio-
Rad, Hercules, CA, USA), separated by SDS-PAGE gels
and transferred onto polyvinylidene difluoride (PVDF)
membranes. For chemiluminecent detection, membranes
were blocked in 5% milk in TBS-T and probed over-
night at 4°C with primary antibodies. Primary antibodies
used were ZEB1 (rabbit polyclonal from Dr. Doug Dar-
ling, University of Louisville, Louisville, KY, USA;
1:1,500 dilution), E-cadherin (clone NCH-38 from
DAKO, Carpinteria, CA, USA; 1 μg/mL), fibronectin
(BD Biosciences, Franklin Lakes, NJ, USA, clone 10/
Fibronectin, 1:5000), moesin (Abcam, Cambridge, MA,
USA, clone EP1863Y, 1:10,000), ERM (Cell Signaling,
Danver, MA, USA, #3142, 1:1000), TrkB (Santa Cruz
Biotechnology, Santa Cruz, CA, USA, H-181, #sc8316,
1:200) and a-tubulin (Sigma-Aldrich, St. Louis, MO,
USA, clone B-5-1-2, 1:30,000). After incubation with
appropriate secondary antibody, results were detected
using Western Lightning Chemiluminescence Reagent
Plus (Perkin-Elmer, Waltham, MA, USA). For fluores-
cent detection, membranes were blocked in 3% BSA
(Sigma-Aldrich) in TBS-T and probed overnight at 4°C
with primary antibodies. Goat anti-rabbit conjugated to
Alexa Fluor 660 (Invitrogen, 1:5,000) and goat anti
mouse conjugated to Alexa Fluor 660 (Invitrogen,
1:5,000) were used as appropriate and signal was
detected by Odyssey (LI-COR, Lincoln, NE, USA).
Wound healing assay
Cells were transfected with miR-200c and controls as
before and 24 hrs later transfected with vectors. Cells were
then plated in six-well plates, allowed to adhere and grow
to confluency. Cells were then treated for two hours with
10 μg/mL mitomycin C (Fisher Scientific, Pittsburgh, PA,
USA). Wounds were made using a p20 pipet tip and cells
were given 24 hrs (Hec50 and BT549) or 48 hrs (AN3CA)
to migrate into wounds. Cells were stained with 0.05%
crystal violet in 6% glutaraldehyde for one hour, rinsed
repeatedly with water, mounted and imaged. For each
condition five representative images were obtained for
quantitation. Quantitation was performed by first thresh-
olding the images to differentiate between cells (black) and
background (white), determining the number of black pix-
els and the number of white pixels and then calculating
the percentage of the image covered by cells.
Anoikis assay (cell viability and cell death ELISA)
Poly-hydroxyethyl methacrylate (poly-HEMA, Sigma-
Aldrich) was reconstituted in 95% ethanol to a concen-
tration of 12 mg/mL. To prepare poly-HEMA coated
plates, 0.5 mL of 12 mg/mL solution was added to each
well of a 24-well plate and allowed to dry overnight in a
laminar flow tissue culture hood. Cells were transfected
as before. Twenty-four hours after transfection 50,000
cells were plated in triplicate in poly-HEMA coated 24-
well plates using regular culture medium. For cell viabi-
lity assay, at 4 and 24 hrs after addition to poly-HEMA
coated plates, viable and dead cells were stained with try-
pan blue and counted using the ViCell cell counter
(Beckman-Coulter, Brea, CA, USA). For cell death ELISA
assay (Roche, San Francisco, CA, USA) cells were plated
as before, but the medium was collected at 2, 4, 8, 24 and
48 hrs post plating. Each sample was pelleted, lysed and
then frozen so that all samples could be read together at
405 nm and 490 nm (reference wavelength). The assay
detects fragmented mono and oligonucleosomes in lysed
cells by first binding histones with a biotinylated antibody
which is bound to a streptavidin-coated plate. Samples
are then bound by an HRP labeled anti-DNA antibody
and color is developed by using an ABTS substrate.
Results
Restoration of miR-200c decreases non-epithelial, EMT
associated genes
We utilize breast and endometrial cancer cell lines in
which we have previously characterized miR-200c levels
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as well as expression of classic epithelial and mesenchy-
mal markers [11,12]. The BT549 and MDA-MB-231 cell
lines are triple negative breast cancer (TNBC) cell lines,
which lack expression of estrogen receptor alpha
(ESR1), progesterone receptors, and HER2/neu. The
TNBC lines lack E-cadherin and express the mesenchy-
mal markers N-cadherin and vimentin and, therefore,
exhibit an EMT phenotype. In contrast, MCF7 cells
represent the luminal A subtype of breast cancer, which
retains epithelial markers including ESR1 and E-cad-
herin. The Hec50 and AN3CA cell lines represent
aggressive type 2 endometrial cancers that have lost
epithelial markers including E-cadherin and ESR1 and
gained mesenchymal markers such as N-cadherin and
vimentin, indicative of EMT. In contrast, Ishikawa cells
represent the less aggressive type 1 endometrial cancer,
which retains epithelial markers and does not express
mesenchymal markers. Transfection of miR-200c mimic
into the dedifferentiated breast and endometrial cancer
lines (BT549, MDA-MB-231, Hec50 and AN3CA)
results in levels of mature miR-200c comparable to
endogenous levels in the more well-differentiated breast
and endometrial cancer lines (MCF7 and Ishikawa) (Fig-
ure 1a). These results indicate that experiments per-
formed using this concentration of mimic result in miR-
200c levels comparable to those observed in cell lines
that have not undergone EMT.
By microarray expression profiling, we previously
identified genes significantly altered upon restoration of
miR-200c to Hec50 cells [12]. Figure 1b is a heatmap of
genes known to be involved in EMT that are statistically
significantly decreased at least 1.5-fold upon restoration
of miR-200c and are bioinformatically predicted to be
targets of miR-200c. The heatmap additionally depicts
miR-200c targets identified by others such as ZEB1 and
2 [8,9], cofilin (CFL1) [9] and WAVE3 [21]. In total we
identified 74 genes that change more than 1.5-fold and
are predicted by two of four target prediction programs
to be direct targets of miR-200c Figure S1 in Additional
file 1. Of these genes, 68 (92%) are repressed and 6 (8%)
are up-regulated when miR-200c is restored. Initial vali-
dation of several of the targets with known involvement
Figure 1 Restoration of miR-200c decreases EMT associated genes. (a) Cells were treated with transfection reagent only (mock), scrambled
negative control (neg) or miR-200c mimic (200c). RNA was harvested after 72 hrs and qRT-PCR was performed for miR-200c. Samples are
normalized to 18S rRNA and presented relative to mock. Columns, mean of three biological replicates, bars, standard deviation of the mean. (b)
Heatmap of genes statistically significantly affected by restoration of miR-200c to Hec50 cells and bioinformatically predicted to be targeted by
miR-200c.
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in EMT revealed that they are down-regulated at the
message level in one or more of our model cell lines
Figure S2 in Additional file 1. Based on these findings,
we selected FN1, MSN, ARHGAP19, LEPR and TrkB
(NTRK2 on the heatmap) to experimentally confirm as
direct targets of miR-200c.
Breast and endometrial cancer cell lines that have
undergone EMT and express ZEB1, also express FN1, MSN
or both
Since there is substantial evidence in the literature for
FN1 and MSN being involved in cancer cell migration,
we assayed the breast and endometrial cancer cell lines
for expression of these proteins (Figure 2). We found
that neither the luminal A breast cancer cell line
(MCF7) or the type 1 endometrial cancer cell line (Ishi-
kawa) express FN1 or MSN, consistent with their pre-
EMT phenotype, indicated by expression of E-cadherin
and lack of ZEB1. In contrast, all of the TNBC and type
2 endometrial cancer lines express either one or both of
these proteins in addition to ZEB1, supporting the
hypothesis that they may play a role in migration in the
absence of miR-200c.
Moesin (MSN), a regulator of cortical actin-membrane
binding, is directly targeted and down-regulated by miR-
200c
MSN connects the actin cytoskeleton and the cell mem-
brane [22] and is strongly up-regulated in cancers with
a poor prognosis, including metastatic breast cancer
[23], where it contributes to migratory and invasive
capacity [24-26]. The 3’ UTR of MSN contains two
putative miR-200c binding sites (Figure 3a) and we
cloned the region containing these sites downstream of
luciferase. When miR-200c is restored, we observe a
37% decrease in luciferase activity only in the presence
of miR-200c and not the controls. To determine the
specificity of this down-regulation, we mutated the puta-
tive miR-200c binding sites and observe that luciferase
activity levels return to levels observed in the absence of
miR-200c; thus, miR-200c binding to these sites specifi-
cally is required for down-regulation. We also observe
that mutating either binding site results in a partial
increase in luciferase activity, but only when both sites
are mutated is there a full restoration of luciferase activ-
ity. Therefore, both binding sites are functional and
required for miR-200c to exert its full effect on the
MSN 3’ UTR. When an antagomiR is used to inhibit
miR-200c binding to the target sites, luciferase activity is
again restored. This indicates that miR-200c specifically
is responsible for targeting the MSN 3’ UTR and the
consequent decrease in luciferase activity. Importantly,
restoration of miR-200c decreases MSN protein levels
(Figure 3b) in two cell lines that express detectable
MSN protein, indicating that direct targeting of MSN by
miR-200c exerts a measurable effect on MSN protein
expression.
Down-regulation of MSN contributes to miR-200c
mediated suppression of migration
Because miR-200c decreases migration, we next sought
to determine the role of MSN in the ability of miR-200c
to inhibit migration. Restoration of miR-200c to BT549
and Hec50 cells results in a dramatic decrease in their
ability to close a wound as indicated by movement of
cells past the initial boundary of the wound (black line)
(Figure 4a). BT549 cells display a 41% decrease in
migratory ability, while Hec50 cells display a 32%
decrease (Figure 4b). The addition of a plasmid encod-
ing MSN lacking its 3’ UTR, rendering it untargetable
by miR-200c, abolishes the ability of miR-200c to
decrease migration (Figure 4a, b) without further
increasing the migratory ability of the mock and nega-
tive control transfected cells. This indicates that miR-
200c targeting of MSN can play a critical role in the
ability of miR-200c to decrease migration in these cell
lines. The levels of MSN protein achieved with the
transfection are reasonable (Figure 4c) and do not inter-
fere with the ability of miR-200c to restore E-cadherin
in these cell lines.
The extracellular matrix protein fibronectin 1 (FN1) is
directly targeted and down-regulated by miR-200c
FN1 is normally expressed by fibroblasts but not epithe-
lial cells, and is a classic marker of the EMT phenotype
Figure 2 Breast and endometrial cancer cells can express FN1
and/or MSN. Breast (a) and endometrial (b) cancer cell lines
analyzed by immunoblot for FN1, MSN, ZEB1, E-cadherin and a-
tubulin expression (loading control).
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and tumorigenicity [27-29]. We [12] and others [8] pre-
viously observed a decrease in FN1 transcript upon
restoration of miR-200c and we sought to determine if
this is due to direct targeting. Like MSN, FN1 contains
two putative miR-200c binding sites in its 3’ UTR.
When miR-200c is restored, we observe a 76% decrease
in luciferase activity only in the presence of miR-200c
and not in the controls (Figure 5a). As for MSN,
mutated constructs show that miR-200c binding to
these sites specifically is required for down-regulation
and both binding sites are functional and required for
miR-200c to exert its full effect on the FN1 3’ UTR.
When an antagomiR is used to inhibit miR-200c binding
to the target sites, luciferase activity is again restored.
This indicates that miR-200c specifically is responsible
for targeting the FN1 3’ UTR and the consequent
Figure 3 Moesin (MSN), a regulator of cortical actin-membrane binding, is directly targeted and down-regulated by miR-200c. (a)
Regions of the 3’ UTR where miR-200c is predicted to bind. Hec50 cells treated with transfection reagent only (mock), scrambled negative
control (neg), miR-200c mimic (200c), miR-200c antagomiR alone (a200c) or in conjunction with miR-200c (a200c + 200c) and luciferase assay
performed. Columns, mean of five replicates, bars, standard deviation of the mean. ANOVA with Tukey-Kramer post-hoc test, ** P < 0.01. (b)
Immunoblot for MSN and a-tubulin (loading control) expression.
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decrease in luciferase activity. Only the AN3CA and
BT549 express detectable protein levels (Figure 2) and
restoration of miR-200c to these cell lines dramatically
decreases FN1 protein expression (Figure 5b).
Down-regulation of FN1 contributes to miR-200c
mediated suppression of migration
We next sought to determine if FN1 plays a role in
miR-200c control of migration. Restoration of miR-200c
to BT549 and AN3CA cells again results in a dramatic
decrease in migration (Figure 6a), which is abrogated by
addition of an untargetable FN1 plasmid. The BT549
cells exhibit a 43% decrease in migratory ability, while
the AN3CA cells decrease 53% (Figure 6b). Thus, down
regulation of FN1 is an additional mechanism by which
miR-200c suppresses migration in aggressive breast and
endometrial cancer cell lines. The levels of FN1 protein
achieved with the plasmid are reasonable and do not
interfere with the ability of miR-200c to restore E-cad-
herin expression in the BT549 cell (Figure 6c). The
Figure 4 Down-regulation of MSN contributes to miR-200c mediated suppression of migration. Cells were transfected with empty vector
(EV) or MSN and 24 hrs later with miRNA constructs. BT549 (left) and Hec50 (right) cells were treated with mitomycin C and given 24 hrs to
migrate. (a) Brightfield images of crystal violet stained cells, dashed black lines indicate edges of the wound immediately after wounding. Scale
bars are 100 μm. (b) Quantitation of migratory ability of cells. Columns, mean of five replicates, bars, standard deviation of the mean. ANOVA, * P
< 0.05, ** P < 0.01, Tukey-Kramer post-hoc test, FF P < 0.01. (c) Immunoblot for MSN, E-cadherin and a-tubulin (loading control).
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AN3CA cells do not re-express E-cadherin following
restoration of miR-200c.
The genes encoding Rho GTPase activating protein 19
(ARHGAP19) and leptin receptor (LEPR) are directly
targeted and down-regulated by miR-200c
ARHGAP19 is a GTPase activating protein that has not
been well characterized, but is predicted to regulate the
activity of Cdc42, RhoA and/or Rac1 [30]. The 3’ UTR
of ARHGAP19 contains one putative miR-200c binding
site. We demonstrate that restoration of miR-200c
causes an 80% reduction in luciferase activity only in the
presence of miR-200c and not in the controls (Figure S3
in Additional file 1). LEPR and its ligand leptin are
involved in the migration/invasion of trophoblasts [31]
and the expression of leptin by mammary epithelial cells
has been linked to tumorigenicity [32-34]. We demon-
strate that restoration of miR-200c causes a 36% reduc-
tion in luciferase activity when the 3’ UTR of LEPR is
placed downstream of luciferase (Figure S4 in Additional
file 1).
The anoikis suppressing neurotrophic receptor tyrosine
kinase 2 (NTRK2 or TrkB) is directly targeted and down-
regulated by miR-200c
TrkB expression leads to anoikis resistance in several
types of cancer, including breast [35-38], and this led us
to investigate the regulation of this cell surface receptor
Figure 5 The extracellular matrix protein fibronectin (FN1) is directly targeted and down-regulated by miR-200c. (a) Regions of the 3’
UTR where miR-200c is predicted to bind. Hec50 cells treated and luciferase assay performed. Columns, mean of five replicates, bars, standard
deviation of the mean. ANOVA with Tukey-Kramer post-hoc test, ** P < 0.01. (b) Immunoblot for FN1 and a-tubulin (loading control) expression.
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by miR-200c. We demonstrate that TrkB is a direct target
of miR-200c, showing a 55% reduction in luciferase activ-
ity (Figure 7a). Luciferase activity is restored following
either mutation of the binding site or addition of an
antagomiR, indicating that miR-200c binds to the 3’ UTR
of TrkB to downregulate it. Additionally, restoration of
miR-200c significantly decreases endogenous TrkB pro-
tein in the BT549 and Hec50 cells (Figure 7b).
miR-200c suppresses anoikis resistance
Given the known role of TrkB in anoikis resistance, we
investigated the effect of miR-200c on anoikis by
Figure 6 Down-regulation of FN1 contributes to miR-200c mediated suppression of migration. Cells were transfected with empty vector
(EV) or FN1 and 24 hrs later with miRNA constructs. BT549 (left) and AN3CA (right) cells were treated with mitomycin C and given 24 or 48 hrs,
respectively, to migrate. (a) Brightfield images of crystal violet stained cells, dashed black lines indicate edges of the wound immediately after
wounding. Scale bars are 100 μm. (b) Quantitation of migratory ability of cells. Columns, mean of five replicates, bars, standard deviation of the
mean. ANOVA, ** P < 0.01, Tukey-Kramer post-hoc test, FF P < 0.01. (c) Immunoblot for FN1, E-cadherin and a-tubulin (loading control).
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performing cell viability assays and cell death ELISAs. In
these assays the cells are plated on poly-HEMA coated
plates, which prevents them from adhering. The cells
are forced to float in suspension for the times indicated
before being harvested for analysis. Cell viability was
determined by trypan blue exclusion and shows that
restoration of miR-200c significantly decreases viability
as quickly as 24 hrs in suspension (Figure 8a). In the
cell death ELISAs, restoration of miR-200c results in an
increase in fragmented nucleosomes, indicating an
increase in apoptosis in these samples (Figure 8b). Thus,
restoration of miR-200c decreases anoikis resistance as
Figure 7 The anoikis suppressing neurotrophic receptor tyrosine kinase 2 (NTRK2 or TrkB) is directly targeted and down-regulated by
miR-200c. (a) The region of the 3’ UTR where miR-200c is predicted to bind. Hec50 cells treated and luciferase assay performed. Columns,
mean of five replicates, bars, standard deviation of the mean. ANOVA with Tukey-Kramer post-hoc test, ** P < 0.01. (b) (Right) Immunoblot for
TrkB and a-tubulin (loading control) expression. (Left) Quantitation of TrkB integrated intensity (I.I.), normalized to a-tubulin and presented
relative to mock. ANOVA, F P < 0.05.
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indicated by a decrease in the viability of suspended
cells and concurrent increase in apoptosis.
Down-regulation of TrkB contributes to miR-200c
mediated suppression of anoikis resistance
To determine if targeting of TrkB is responsible for the
ability of miR-200c to restore sensitivity to anoikis, we
used a plasmid encoding TrkB lacking the 3’ UTR, ren-
dering it untargetable by miR-200c. Restoration of miR-
200c enhances sensitivity to anoikis (Figures 8 and 9),
but this phenotype is completely reversed in the pre-
sence of exogenous, untargetable TrkB (Figure 9a, c).
However, it is important to note that the addition of
exogenous TrkB does not decrease the amount of cell
Figure 8 miR-200c increases sensitivity to anoikis. Breast (left) and endometrial (right) cancer cells were transfected with miRNA constructs
and plated on poly-HEMA coated plates. Cells were collected for viability analysis by trypan blue exclusion (a) or apoptosis analysis by cell death
ELISA (b). Columns, mean of three biological replicates, bars, standard deviation of the mean. ANOVA, * P < 0.05, ** P < 0.01.
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death in mock or negative control transfected cells. This
indicates that miR-200c targeting of TrkB plays a critical
role in the ability of miR-200c to reverse anoikis
resistance.
Discussion
Progression and metastasis of carcinomas is a multistep
process. EMT is thought to aid cancer cells as they
invade through basement membrane and stroma,
intravasate into blood or lymph vessels, and may also
facilitate anoikis resistance, allowing tumor cells to sur-
vive the journey to the metastatic site. We sought to
identify additional direct targets of miR-200c that med-
iate its potent effects.
Three of the new direct targets of miR-200c that we
identify, MSN, FN1, and ARHGAP19, have been impli-
cated in migration and invasion. MSN localizes to the
trailing edge of invasive melanoma cells and disruption
Figure 9 Down-regulation of TrkB contributes to miR-200c mediated suppression of anoikis resistance. Cells were transfected with
empty vector (EV) (left) or TrkB (right) and 24 hrs with miRNA constructs. Twenty-four hours later cells were plated on poly-HEMA coated plates
and cell death ELISA performed at time points indicated (a) and (c). Columns, mean of three biological replicates, bars, standard deviation of the
mean. ANOVA, * P < 0.05, ** P < 0.01. (b) and (d) Immunoblot for TrkB and a-tubulin (loading control).
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of this localization leads to decreased metastasis [25].
MSN expression correlates with poor prognosis in oral
squamous cell carcinoma [24] and basal breast cancer
[23], a subtype with high risk of metastasis and recur-
rence. FN1 functions in cell migration through integrin
binding [39] and can activate focal adhesion kinase
(FAK) leading to increased motility and invasion of car-
cinoma cells [27,28]. ARHGAP19 is a member of a
family of GTPase activating proteins, and other family
members, 8, 9, 12 and 15, are expressed in several types
of cancer and activate Cdc42, Rac1 or RhoA [40-43],
small GTPases required for migration. We demonstrate
that FN1 and MSN are, at least in some cell lines, criti-
cal targets sufficient to mediate miR-200c’s ability to
inhibit migration in an in vitro wound healing assay. In
some cell lines both MSN and FN1 are expressed, and
in those cells both MSN and FN1 may contribute to
migratory potential, but they are both repressed when
miR-200c is restored. In other TNBC cells and type 2
endometrial cancer cells, either MSN or FN1 are
expressed but not both. It is possible that even though
miR-200c is absent, additional miRNA(s) that target
these genes may be retained in some cells, or alterna-
tively, factors that induce these genes at the promoter
may be differentially expressed. In some cases ARH-
GAP19 may additionally contribute to migratory capa-
city; however, at present there is no antibody available
to detect this protein. Loss of miR-200c could permit
any of these genes, typically expressed in the more
motile mesenchymal or neuronal cell types, to be inap-
propriately translated and expressed in epithelial cells.
Expression of proteins such as MSN that actively contri-
bute to cell motility by promoting front-rear polarity,
combined with the loss of E-cadherin (which would
decrease cell-cell attachments and reduce apical-basal
polarity), may significantly contribute to the invasive
capacity of carcinomas.
We demonstrate that restoration of miR-200c leads
to a dramatic increase in sensitivity to anoikis (over a
100% increase in anoikis in some cell lines) and iden-
tify TrkB as a novel direct target of miR-200c. TrkB is
a tyrosine kinase cell surface receptor typically
expressed on neurons, which can be inappropriately
expressed in carcinomas [44]. In breast and ovarian
cancer cell lines TrkB induces anoikis resistance
[31,33] and can induce EMT through activation of
Twist [41]. We previously demonstrated that miR-200c
does not affect apoptosis when endometrial cancer
cells are attached to plastic, although it does enhance
apoptosis induced by taxanes [11,12]. Thus, we con-
clude that miR-200c specifically enhances anoikis sen-
sitivity, suggesting that restoration of miR-200c could
limit the ability of breast and endometrial cancer cells
to survive in the bloodstream.
Interestingly, all of the new miR-200c direct targets
that we identify in this study (as well as other previously
identified targets such as ZEB1/2 and TUBB3) contri-
bute to the designation of this miRNA as a “guardian of
the epithelial phenotype” because they are genes typi-
cally expressed in cells of mesenchymal or neuronal ori-
gin, but not in normal, well-differentiated epithelial cells.
Not all of the target genes that we identify change at
the message level upon restoration of miR-200c. For
example, although miR-200c directly targets ARHGAP19
(Figure S3 in Additional file 1), the message is down-
regulated by addition of miR-200c in only 3 of 4 cell
lines (Figure S2 in Additional file 1). There are several
possible explanations for interference between a miRNA
and its mRNA target in some cell lines. The miR-200c
target site may be mutated or absent due to a shorten-
ing of the 3’ UTR [46-49] or there may be RNA binding
proteins present in particular cell lines that prevent
miR-200c from binding [50]. Importantly, for all of the
targets that we follow up on in this study (MSN, FN1
and TrkB), protein levels are affected by miR-200c, indi-
cating that it does have an affect on translation of these
genes, regardless of whether it also affects degradation
of the message.
Conclusions
In summary, miR-200c inhibits migration and invasion
[9-13], stemness [51,52], and chemoresistance [11,12]
and we now identify a completely novel role for miR-
200c - the ability to reverse anoikis resistance, an impor-
tant additional step in the metastatic cascade. We iden-
tify new targets of miR-200c, which together with
previously identified targets, comprise a program of
genes normally restricted to cells of mesenchymal or
neuronal origin. We specifically pinpoint MSN and FN1
as well as TrkB as targets that can respectively mediate
the ability of miR-200c to inhibit cell motility and anoi-
kis resistance.
Members of the miR-200 family are down-regulated in
breast cancer stem cells and normal mammary gland
stem cells [51]. Polycomb complexes facilitate stem cell
self-renewal and pluripotency, and both Bmi1, a compo-
nent of the PRC1 polycomb complex, and Suz12, a com-
ponent of the PRC2 polycomb complex, have been
identified as targets of miR-200 family members [51-53].
It is interesting to speculate as to whether expression of
TrkB is involved in the ability of cancer stem cells to
resist anoikis.
If feasible, effective in vivo delivery of miR-200c could
potentially inhibit multiple steps in tumor progression,
including tumor formation, cell motility/invasiveness,
anoikis resistance and chemoresistance, by virtue of
simultaneously repressing multiple, yet specific, targets
expressed in carcinoma cells exhibiting an EMT
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phenotype. Although one in vivo study demonstrated
that introduction of miR-200c reduced the ability of pri-
mary human breast cancer stem cells to form tumors in
immune compromised mice [51], further in vivo studies
will be necessary to specifically isolate the effects of
miR-200 on other steps in the metastatic cascade, such
as its potential to reverse anoikis resistance.
Additional material
Additional file 1: Additional experimental data and the sequences
of primers used in cloning and qRT-PCR.
Abbreviations
DMEM: Dulbecco’s modified eagle’s medium; EMT: epithelial to
mesenchymal transition; ESR1: estrogen receptor alpha; FAK: focal adhesion
kinase; FBS: fetal bovine serum; FN1: fibronectin 1; LEPR: leptin receptor;
MSN: moesin; NEAA: non-essential amino acids; poly-HEMA: poly-
hydroxyethyl methacrylate; TNBC: triple negative breast cancer; UTR:
untranslated region.
Acknowledgements
We thank Daniel Peeper (Netherlands Cancer Institute) for his generous gift
of pBabe-TrkB. We thank the development teams for the open source
software packages Graphical Image Manipulation Program (GIMP) and
Zotero. We acknowledge Christopher Korch, Ph.D., in the University of
Colorado Cancer Center DNA Sequencing and Analysis Core (supported by
the NIH/National Cancer Institute Cancer Core Support Grant P30 CA046934)
for sequencing of constructs and verification of cell line identity. This work
was supported by the Department of Defense Breast Cancer Research
Program Idea Award BC084162 and Susan G Komen Foundation KG090415
(JK Richer).
Author details
1Program in Cancer Biology, Department of Pathology, University of
Colorado, Anschutz Medical Campus, Mail Stop 8104, P.O. Box 6511, Aurora,
CO, USA.2Department of Pathology, University of Colorado, Anschutz
Medical Campus, Mail Stop 8104, P.O. Box 6511, Aurora, CO, USA.
Authors’ contributions
ENH performed experimental studies. DRC performed array profiling studies
and generated the heatmap in Figure 1. All authors contributed intellectual
input towards the design, implementation, and interpretation of results. ENH
and JRK drafted the manuscript and all authors read and approved the final
manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 22 October 2010 Revised: 7 March 2011
Accepted: 18 April 2011 Published: 18 April 2011
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doi:10.1186/bcr2867
Cite this article as: Howe et al.: Targets of miR-200c mediate
suppression of cell motility and anoikis resistance. Breast Cancer Research
2011 13:R45.
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