Epithelial–mesenchymal transition in colorectal cancer metastasis: A system review

Abstract

Tumor metastasis is a multi-step process by which tumor cells disseminate from their primary site and form secondary tumors at a distant site. And metastasis is the major cause of death in the vast majority of cancer patients. However, the mechanisms underlying each step remain obscure. In the past decade, a developmental program epithelial-to-mesenchymal transition (EMT) has been increasingly recognized to play pivotal and intricate roles in promoting carcinoma invasion and metastasis. The EMT process is very complex and controlled by various families of transcriptional regulators through different signaling pathways. In this system review, we focus on the molecular network of the EMT program and its malignant phenotypes associated with metastasis in colorectal cancer (CRC), including cancer stem cells, tumor budding, circulating tumor cells and drug resistance. A better understanding of the molecular regulation of the dynamic EMT program during tumor metastasis will help to provide much-needed therapeutic interventions to target this program when treating metastatic CRC.
Copyright © 2015 Elsevier GmbH. All rights reserved.

Full text

Pathology
–
Research
and
Practice
211
(2015)
557–569
Contents
lists
available
at
ScienceDirect
Pathology
–
Research
and
Practice
jou
rn
al
hom
epage:
www.elsevier.com/locate/prp
Review
Epithelial–mesenchymal
transition
in
colorectal
cancer
metastasis:
A
system
review
Hui
Caoa,b,
Enping
Xua,b,
Hong
Liua,c,
Ledong
Wana,b,
Maode
Laia,b,∗
aDepartment
of
Pathology,
School
of
Medicine,
Zhejiang
University,
Hangzhou
310058,
China
bKey
Laboratory
of
Disease
Proteomics
of
Zhejiang
Province,
Hangzhou
310058,
China
cZhejiang
Normal
University-Jinhua
People’s
Hospital
Joint
Center
for
Biomedical
Research,
Jinhua
321004,
China
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
23
April
2015
Accepted
20
May
2015
Keywords:
Metastasis
Epithelial–mesenchymal
transition
Molecular
related
network
Malignant
phenotypes
Colorectal
cancer
a
b
s
t
r
a
c
t
Tumor
metastasis
is
a
multi-step
process
by
which
tumor
cells
disseminate
from
their
primary
site
and
form
secondary
tumors
at
a
distant
site.
And
metastasis
is
the
major
cause
of
death
in
the
vast
majority
of
cancer
patients.
However,
the
mechanisms
underlying
each
step
remain
obscure.
In
the
past
decade,
a
developmental
program
epithelial-to-mesenchymal
transition
(EMT)
has
been
increasingly
recognized
to
play
pivotal
and
intricate
roles
in
promoting
carcinoma
invasion
and
metastasis.
The
EMT
process
is
very
complex
and
controlled
by
various
families
of
transcriptional
regulators
through
different
signaling
pathways.
In
this
system
review,
we
focus
on
the
molecular
network
of
the
EMT
program
and
its
malignant
phenotypes
associated
with
metastasis
in
colorectal
cancer
(CRC),
including
cancer
stem
cells,
tumor
budding,
circulating
tumor
cells
and
drug
resistance.
A
better
understanding
of
the
molecular
regulation
of
the
dynamic
EMT
program
during
tumor
metastasis
will
help
to
provide
much-needed
therapeutic
interventions
to
target
this
program
when
treating
metastatic
CRC.
©
2015
Elsevier
GmbH.
All
rights
reserved.
Contents
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Introduction
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2.
EMT
regulation
in
colorectal
cancer
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2.1.
Transcriptional
regulation
of
EMT
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2.2.
EMT-related
signaling
pathways
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2.3.
Non-coding
RNAs
in
EMT
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2.4.
Microenvironmental
regulation
of
EMT
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562
3.
Malignant
phenotypes
of
EMT
in
colorectal
cancer
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563
3.1.
EMT
and
tumor
budding
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563
3.2.
EMT
and
drug
resistance
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563
4.
Cancer
therapeutics
targeting
EMT
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564
5.
Conclusions
and
future
perspectives
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564
Acknowledgements
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565
References
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565
1.
Introduction
Colorectal
cancer
(CRC)
is
the
third
most
commonly
diagnosed
cancer
and
the
fourth
most
common
cause
of
cancer-related
death
∗Corresponding
author
at:
Department
of
Pathology,
School
of
Medicine,
Zhejiang
University,
Hangzhou
310058,
China.
Tel.:
+86
571
88208200;
fax:
+86
571
88208197.
E-mail
address:
lmp@zju.edu.cn
(M.
Lai).
globally,
accounting
for
over
1.2
million
new
cases
and
608,700
deaths
per
year
[1].
Although
improved
treatment
strategies
involving
surgery
and
chemo-
and
radio-therapy
have
increased
the
overall
survival
rates
in
the
early
stages,
40–50%
of
all
patients
with
CRC
present
with
metastasis
either
at
the
time
of
diagnosis
or
as
recurrent
disease
upon
intended
curative
therapy
[2].
Most
CRC
patients
with
distant
metastasis
are
not
suitable
candidates
for
conventional
therapy
and
exhibit
a
poor
5-year
survival
rate
of
<10%
[3,4].
Given
this,
a
better
understanding
of
the
molecu-
lar
mechanisms
underlying
local
invasion
and
distant
metastasis
http://dx.doi.org/10.1016/j.prp.2015.05.010
0344-0338/©
2015
Elsevier
GmbH.
All
rights
reserved.

558
H.
Cao
et
al.
/
Pathology
–
Research
and
Practice
211
(2015)
557–569
Fig.
1.
Model
of
tumor
metastasis
over
time.
Epithelial
cells
undergoing
genetic
transformation
become
a
carcinoma
in
situ.
In
response
to
EMT-promoting
signals,
a
subpopulation
of
tumor
cells
at
the
invasive
front
may
lose
epithelial
properties
and
invade
the
local
matrix
(step
I)
and
enter
into
the
vasculature
(step
II).
Cancer
cells
traveling
through
the
circulation
are
circulating
tumor
cells
(CTCs).
These
cells
that
have
undergone
EMT
are
then
transported
in
the
system
circulation
and
survive
via
various
mechanisms
(step
III).
At
a
distant
tissue
site,
the
mesenchymal
phenotype
facilitates
tumor
cell
extravasation
into
the
parenchyma
(step
IV)
to
establish
micrometastases.
This
initial
seeding
of
tumor
cells
at
distant
sites
can
occur
rapidly,
after
which
cells
may
enter
a
prolonged
dormancy.
Subsequent
colonization
of
distant
organs
requires
the
reversion
of
the
EMT
and/or
activation
of
the
MET
program
and
the
cells
gradually
re-acquire
epithelial
traits
such
as
rapid
proliferation
to
establish
macroscopic
tumors
(step
V).
In
certain
tumor
types,
the
layout
of
the
circulation
may
be
the
strongest
determinant
of
metastatic
tropism,
such
as
the
behavior
of
colorectal
cancer
(CRC),
which
has
a
strong
preference
for
generating
liver
metastases.
is
imperative
to
facilitate
the
development
of
effective
therapeutic
strategies
for
patients
with
metastatic
CRC,
or
to
prevent
incipient
metastasis
in
the
adjuvant
setting.
Tumor
metastasis
consists
of
sequential,
interlinked,
and
selec-
tive
steps
[5],
and
many
of
the
steps
are
favored
by
conversions
between
two
cellular
states
–
the
epithelial
and
mesenchymal
phe-
notypes
(Fig.
1).
The
“epithelial–mesenchymal
transition”
(EMT),
a
key
developmental
regulatory
program,
has
been
reported
to
play
critical
and
intricate
roles
in
promoting
tumor
invasion
and
metastasis
in
epithelium-derived
carcinomas
in
recent
years.
The
EMT
program
allows
stationary
and
polarized
epithelial
cells,
which
are
connected
laterally
via
several
types
of
junctions
and
normally
interact
with
the
basement
membrane
via
their
basal
surfaces
to
maintain
apical–basal
polarity,
to
undergo
multi-
ple
biochemical
changes
that
enable
them
to
disrupt
cell–cell
adherence,
lose
apical–basal
polarity,
dramatically
remodel
the
cytoskeleton,
and
acquire
mesenchymal
characteristics
such
as
enhanced
migratory
capacity,
invasiveness,
elevated
resistance
to
apoptosis
and
greatly
increased
production
of
ECM
components
[6].
Mesenchymal–epithelial
transition
(MET),
the
reversal
of
EMT,
seems
to
occur
after
dissemination
and
the
subsequent
formation
of
distant
metastases
[7].
However,
research
on
the
mechanism
underlying
the
MET
is
limited
and
needs
further
investigation.
The
EMT
process
has
been
reported
in
multiple
epithelial
cancers
including
breast
[8],
prostate
[9]
and
colorectal
cancer
[10].
This
review
focuses
on
recent
insights
into
the
control
of
EMT
at
different
molecular
levels
and
their
conceptual
implications
for
CRC
metastasis,
including
reflections
on
whether
and
how
the
EMT
can
be
considered
as
an
applicable
target
for
therapeutic
strategies
to
fight
CRC.
2.
EMT
regulation
in
colorectal
cancer
2.1.
Transcriptional
regulation
of
EMT
Accumulating
evidence
has
confirmed
that
the
EMT
plays
a
prominent
and
complex
role
in
human
CRC.
Downregulated
E-cadherin
expression
indicates
the
presence
of
lymph
node
metas-
tases,
poor
tumor
differentiation
and
a
worse
prognosis
in
CRC
patients
[11,12],
consistent
with
the
role
of
E-cadherin
as
a
gate-
keeper
of
the
epithelial
state
in
carcinomas
[13,14].
Conversely,
increased
expression
of
vimentin
is
significantly
associated
with
lymph
node
metastasis
and
poor
prognosis
[15].
The
EMT
process
is
initially
driven
by
three
core
groups
of
transcriptional
regulators,
which
are
able
to
suppress
E-cadherin
transcription
directly
or
indirectly.
The
first
group
is
the
tran-
scription
factors
(TFs)
of
the
Snail
zinc-finger
family,
including
SNAI1
and
SNAI2
(SLUG)
[16–18].
The
second
group
is
the
distantly
related
zinc-finger
E-box-binding
homeobox
family
proteins
ZEB1
and
ZEB2
(SIP1)
[19,20].
The
last
group
is
the
basic
helix–loop–helix
(bHLH)
family
of
transcription
factors,
including
TWIST1,
TWIST2
and
E12/E47
[21–23].
In
CRC,
85%
of
resected
specimens
have
mod-
erate
to
strong
Twist1
expression,
which
is
notably
more
than
either
SNAI1
or
SLUG
[24–27].
SLUG
and
ZEB1
expression
is
sig-
nificantly
correlated
with
lower
expression
of
E-cadherin
[25,28]
and
up-regulation
of
ZEB1
and
ZEB2
at
the
invasion
front
both
cor-
relate
with
shorter
survival
times
[29,30].
Otherwise,
ZEB1
can
also
activate
the
transcription
of
the
urokinase
plasminogen
activator
(uPA)
involved
in
EMT-related
degradation
of
ECM
and
inhibit
PAI-
1,
an
inhibitor
of
uPA
[31].
Furthermore,
a
significant
correlation
has
been
reported
between
SLUG
and
vimentin
expression,
and
upreg-
ulation
of
SLUG
has
emerged
as
an
independent
prognostic
factor
and
a
predictive
marker
of
lymph
node
metastasis
and
sprouting
angiogenesis
in
CRC
patients
[15,32].
Using
TWIST1
immunostain-
ing
in
combination
with
fluorescence
in
situ
hybridization
(FISH)
analysis
of
the
chromosome
translocation
unique
to
human
CRC
cells,
it
has
been
found
that
17
out
of
20
colon
tumors
contain
TWIST1+tumor
cells
with
a
mesenchymal
phenotype
[33].
In
CRC
patients,
the
TWIST1
overexpression
is
associated
with
nodal
inva-
sion,
male
sex
and
unfavorable
outcomes
[27,34,35].
More
recently,
emerging
evidence
has
indicated
that
many
other
TFs
are
involved
in
the
EMT
and
CRC
progression.
They
belong
to
different
families
including
Brachyury
[36,37],
AP4
[38],

H.
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et
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/
Pathology
–
Research
and
Practice
211
(2015)
557–569
559
Table
1
Molecular
factors
involved
in
EMT
and
CRC
progression:
transcriptional
regulation.
Molecules
Involvement
in
family
Clinical
significance
in
CRC
Refs.
Transcription
factors:
direct
binding
to
the
CDH1
promoter
SNAI1
Zinc-finger
protein Metastasis,
chemoresistance,
poor
prognosis [24,175]
SLUG
Zinc-finger
protein
Metastasis,
poor
survival,
poor
prognosis,
pathological
angiogenesis,
drug
resistance
[15,25,32,176]
ZEB1
Zinc-finger
E-box
binding
homeobox
protein
Invasion,
metastasis,
poor
survival
[28,29]
ZEB2
Zinc-finger
E-box
binding
homeobox
protein
Tumor
progression,
poor
survival
[30]
Brachyury
T-box
family
of
transcription
factor
Poor
prognosis
[36,37]
AP4
bHLH-zipper
factor
Liver
metastasis,
poor
survival
[38]
Other
transcription
factors
associated
with
EMT
TWIST1
bHLH
factor
Nodal
invasion,
poor
prognosis,
poor
survival
[33–35]
FOXC2
Forkhead
family
of
transcription
factor
Lymph
node
metastasis
[39,40]
TCF4
Class
I
bHLH
factor
ND
[41]
SOX2
SRY-related
HMG-box
(SOX)
factor
Liver
and
lymph
node
metastasis
[42]
OCT4
Octamer-binding
transcription
factor
Liver
metastasis
[43]
Nanog
Homebox
protein
Lymph
node
metastasis,
poor
prognosis
[44]
PROX1
Homebox
protein
Advanced
tumor
staging,
lymph
node
metastasis
[45]
SIX1
Homebox
protein ND
[46]
PRRX1
Homebox
protein
Metastasis
and
poor
prognosis
[47]
HMGA1
High-mobility
group
protein
ND
[48]
Fra-1
Fos
family
of
transcription
factor
Poor
recurrence-free
survival,
a
higher
T-stage
[49]
Transcriptional
coregulators
FHL2
LIM-only
protein,
SNAI1-FHL2
Invasion,
metastasis
[51,52]
BRG1
SWI/SNF
chromatin-remodeling
protein,
ZEB1-BRG1
ND
[53,54]
SP1
SP/KLF
family
of
transcription
factor,
ZEB2-SP1 ND
[55]
P21
Cyclin-dependent
kinase
inhibitor,
ZEB1-P21
ND
[56]
Abbreviations:
EMT,
epithelial-mesenchymal
transition;
CRC,
colorectal
cancer;
ND,
not
determined;
FOXC2,
forkhead
box
C2;
SOX2,
SRY
(sex
determining
region
Y)-box
2;
OCT4,
octamer-binding
transcription
factor
4;
PROX1,
prospero
homeobox
1;
SIX1,
SIX
homeobox
1;
PRRX1,
paired
related
homeobox
1;
HMGA1,
High-mobility
group
A1;
ZNF281,
zinc
finger
protein
281;
FHL2,
four
and
a
half
LIM
protein
2.
fork-head
box
protein
C2
(FOXC2)
[39,40],
E2-2
(also
known
as
TCF4)
[41],
SOX2
[42],
OCT4
[43],
Nanog
[44],
prospero
homeobox
1
(PROX1)
[45],
homeobox
protein
SIX1
[46],
paired
related
home-
obox
1
(PRRX1)
[47],
HMGA1
[48],
Fra-1
[49],
and
ZNF281/ZBP99
[50].
Apart
from
Brachyury
and
AP4,
the
other
TFs
seem
to
trigger
the
EMT
without
direct
binding
to
the
CDH1
promoter.
In
addition,
other
factors
negatively
regulate
E-cadherin
through
interaction
with
core
EMT–TFs,
such
as
four
and
a
half
LIM
protein
2
(FHL2)
[51,52],
SP1
[53,54],
BRG1
[55],
and
p21
[56].
Regardless
of
the
mechanism,
E-cadherin
repressors
function
as
full
EMT
inducers
in
many
cellular
contexts,
regulating
the
expression
of
various
molecules
that
repress
the
epithelial
character
and
promote
the
mesenchymal
state
(Table
1).
2.2.
EMT-related
signaling
pathways
Carcinoma
cells
are
thought
to
undergo
the
EMT
in
response
to
a
combination
of
extracellular
signals
in
their
microenvironment.
All
major
development
signaling
circuits,
including
TGF-␤,
Wnt,
and
growth
factor
receptor
signaling
cascades,
have
been
implicated
in
some
aspects
of
the
EMT
program
and
play
roles
in
the
progression
of
various
cancers
[57],
including
CRC
(Fig.
2).
The
TGF-␤/Smad
signaling
pathway
plays
an
important
and
heterogeneous
role
in
CRC
and
is
an
essential
driver
of
the
EMT
[58,59].
TGF-␤
ligands
cause
dimerization
of
the
T␤RI
and
T␤RII
receptors
in
the
membrane,
which
leads
to
the
phosphorylation
of
Smad
proteins
[60,61].
Activated
Smad2
and
Smad3
localize
to
the
nucleus
with
Smad4
to
serve
as
a
transcriptional
regulator
[62].
Mutational
inactivation
of
the
TGF-␤
pathway
is
a
key
reason
for
CRC
progression.
Alterations
in
TGF-␤
receptors
and
Smad
signal-
ing
are
first
detected
in
advanced
adenomas
and
affect
40–50%
of
all
CRC
[63,64].
Loss
of
Smad4
occurs
in
up
to
30%
metastatic
CRCs
and
correlates
highly
significantly
with
the
loss
of
E-cadherin
and
increased
levels
of
␤-catenin
[65,66].
In
Smad4-null
cell
lines,
TGF-␤
induces
invasion,
migration,
tumorgenesis
and
the
potential
for
metastasis,
while
incubation
with
LY2109761
(a
TGF-␤-
receptor
kinase
inhibitor)
reverses
these
effects
and
produces
MET
properties
[67].
The
underlying
mechanism
may
be
a
corresponding
increase
in
MMP9
enhanced
by
hypoxia-induced
Glut1
expression
[68].
Similarly,
Smad4
suppresses
invasiveness
and
mediates
the
reversion
of
SW480
cells
from
a
mesenchymal-like
phenotype
to
a
polarized
epithelial
phenotype;
furthermore
Smad4
reconstitution
is
associated
with
the
downregulation
of
endogenous
TGF-␤,
sug-
gesting
that
autocrine
TGF-␤
signaling
may
be
involved
in
the
EMT
[69].
Smad4
is
also
a
negative
regulator
of
STAT3
activation
[70];
conversely,
loss
of
Smad4
leads
to
aberrant
activation
of
STAT3,
which
may
directly
contribute
to
the
EMT
process
and
ZEB1
expres-
sion
in
CRC
progression
[71].
This
absence
of
Smad4
expression
represents
a
promising
new
biomarker
to
predict
liver
metastasis
in
CRC
patients
[72].
Most
interestingly,
Smad4
is
also
a
central
component
of
bone
morphogenetic
protein
(BMP)
signaling
path-
way.
The
loss
of
Smad4
causes
BMP
signaling
to
switch
from
tumor
suppressive
to
metastasis
promoting
by
triggering
EMT
and
activa-
tion
of
Rho
and
ROCK
[73].
Taken
together,
these
findings
suggest
that
loss
of
Smad4
may
underlie
the
functional
shift
of
TGF-␤
and
BMP
from
a
tumor
suppressor
to
a
tumor
promoter.
In
addition,
fucosylated
TGF-␤
receptors
are
able
to
transduce
a
signal
for
EMT
in
CRC
cells
[74].
Wnt
signaling
is
also
an
important
contributor
to
CRC
progres-
sion
and
regulators
of
the
EMT
[75].
Aberrant
activation
of
canonical
Wnt
signaling
is
pathognomonic
of
CRC
harboring
functional
muta-
tions
in
either
APC
or
␤-catenin
[64,76].
Indeed,
increased
nuclear
␤-catenin
is
found
in
dedifferentiated
mesenchyme-like
tumor
cells
undergoing
an
active
EMT
at
the
invasive
front
accompanied
by
changes
in
E-cadherin
expression
[77,78],
and
then
the
␤-
catenin/TCF4
complex
transcriptional
activates
ZEB1
directly
and
upregulates
MT1–MMP9
and
LAMC2
which
have
been
linked
to
invasiveness
in
CRC
[41].
Conversely,
upregulation
of
dickkopf-1
(DKK1),
a
potent
inhibitor
of
Wnt
signaling,
suppresses
the
CRC
progression
by
inhibiting
the
EMT
program
[79].
Most
intriguingly,
Axin2,
a
canonical
Wnt
suppressor,
acts
as
a
potent
tumor
pro-
moter
by
up-regulating
the
activity
of
SNAI1
to
induce
a
functional
EMT
program
and
drive
metastatic
activity
both
in
vitro
and
in
vivo
[80].
In
turn,
SNAI1
promotes
Wnt
target
genes
and
interacts
with

560
H.
Cao
et
al.
/
Pathology
–
Research
and
Practice
211
(2015)
557–569
Fig.
2.
Simplified
overview
of
signaling
networks
and
tumor
microenvironment
that
regulate
EMT
in
CRC.
The
epithelial–mesenchymal
transition
(EMT)
process
is
regulated
by
signaling
pathways
that
co-operate
to
induce
the
full
EMT
response.
Transforming
growth
factor-␤
(TGF-␤),
Wnt,
and
receptor
tyrosine
kinases
(RTKs)
can
induce
an
EMT
through
multiple
different
signaling
circuits,
and
the
relative
importance
of
each
of
these
may
depend
on
the
particular
cellular
context.
TGF-␤
induces
phosphorylation
of
Smad
proteins
and
activated
Smad2
and
Smad3
localize
to
the
nucleus
with
Smad4
to
transactivate
SNAI1
expression.
Smad4
is
an
important
negative
regulator
in
this
pathway
and
also
suppresses
STAT3
activation,
which
may
directly
contribute
to
the
EMT
process
and
ZEB1
expression.
Wnt
signaling
promotes
EMT
by
inhibiting
glycogen
synthase
kinase-3␤
(GSK3␤)
to
stabilize
␤-catenin,
which
translocates
to
the
nucleus
to
engage
the
TCF4
that
transcriptionally
activates
ZEB1
and
SNAI1
directly.
In
turn,
SNAI1
promotes
Wnt
target
genes
and
interacts
with
␤-catenin.
Dickkopf-1
(DKK1),
a
potent
inhibitor
of
LRP5/6,
inhibits
the
EMT
program.
Axin2,
a
canonical
Wnt
suppressor,
acts
as
a
potent
tumor
promoter
by
up-regulating
the
activity
of
SNAI1
and
inducing
a
functional
EMT
program.
Several
growth
factors
that
act
through
RTKs,
including
epidermal
growth
factor
(EGF),
fibroblast
growth
factor
(FGF),
and
hepatocyte
growth
factor
(HGF),
can
induce
an
EMT.
The
RAS-RAF-MEK1/2-ERK1/2
MAPK
signaling
cascade
is
a
major
pathway
activated
by
RTKs
in
response
to
growth
factors.
Egr-1
and
Fra-1,
an
AP-1
protein,
are
responsive
for
MEK1-induced
SNAI1
and
SLUG
expression,
respectively.
Raf-1
kinase
inhibitor
protein
(RKIP)
and
PHLPP
are
negative
regulators
by
inhibiting
RAF.
RTK-activated
AKT
can
induce
SNAI1
expression
through
nuclear
factor-␬B
(NF-␬B)
and
stabilize
SNAI1
and
␤-catenin
by
inhibiting
GSK3␤,
thus
cooperating
with
Wnt
signaling.
PTEN
is
a
negative
regulatory
factor
that
antagonizes
the
PI3K
signaling
pathway.
The
tumor
environment
is
a
complex
network
of
inflammatory
and
immune
cells,
carcinoma-associated
fibroblasts
(CAFs),
mesenchymal
stem/stromal
cells
(MSCs),
normal
epithelial
cells,
endothelial
cells
and
hypoxia,
soluble
factors,
signaling
molecules
and
extracellular
matrix
components.
IL-6
is
an
important
cytokine
in
the
microenvironment.
The
IL-6/IL-6R/STAT3/miR-34a
feedback
loop
with
respect
to
EMT
contributes
to
CRC
metastasis.
␤-catenin;
this
indicates
positive
feedback
stimulation
of
the
Wnt
pathway
by
activation
of
SNAI1
[81].
Several
results
indicate
the
effects
of
the
RAS/ERK1/2
path-
ways
in
the
EMT
of
CRC.
First
of
all,
oncogenic
RAS
overexpression
induces
EMT
in
Caco2
cells
by
affecting
histone
covalent
modifica-
tions
to
regulate
CyclinD1
and
E-cadherin
[82].
SLUG
is
regulated
by
the
RAS
pathway
and
may
be
a
target
for
treatment
of
the
broad
spectrum
of
CRC
with
mutant
RAS
that
has
undergone
EMT
[83].
Further,
it
has
been
demonstrated
that
the
BRAF
and
RAS
oncogenes
regulate
Rho
GTPases
to
mediate
cell
migration
and
invasion
along
with
EMT-like
properties
alone
or
in
cooperation
with
the
TGF-␤
pathway
[84].
In
intestinal
epithelial
cells,
consti-
tutively
active
MEK1
is
enough
to
induce
an
EMT
associated
with
tumor
invasion
and
metastasis.
In
brief,
Egr-1
and
Fra-1,
an
AP-1
protein,
are
responsive
to
MEK1-induced
SNAI1
and
SLUG
expres-
sion,
respectively
[85].
Meanwhile,
AP1
is
induced
by
the
activation
of
ERK/AKT
pathways
and
then
activates
EZH2
expression,
which
controls
the
EMT
by
targeting
integrin
␣2
[86].
Loss
of
RKIP
(Raf-
1
kinase
inhibitor
protein)
correlates
with
NF-␬B
activation
and
loss
of
E-cadherin
and
predicts
frequent
distant
metastasis
[87].
PHLPP,
a
negative
regulator
of
RAF1
and
RAF/MEK/ERK
signaling,
can
reduce
CRC
cell
motility
and
tumor
progression
in
mice
by
sup-
pressing
EMT.
However,
it
is
downregulated
in
samples
from
CRC
patients
and
point
mutations
in
PHLPP
genes
has
been
identified
in
2–3%
of
tumors
[88].
Activation
of
PI3K/AKT
signaling
though
mutations
in
PIK3CA
or
loss
of
PTEN
is
associated
with
CRC
progression
[89].
The
potential
role
of
AKT
signaling
in
regulating
EMT
and
angiogenesis
in
CRC
is
to
elevate
the
expression
of
important
EMT-TFs
(e.g.,
SNAI1
and
SLUG)
both
in
vitro
and
in
vivo
[90].
PRL-3,
a
metastasis-associated
phosphatase,
plays
an
initiating
role
to
trigger
the
EMT
switch
dur-
ing
cancer
metastasis
by
activating
AKT
and
inactivating
PTEN
[91].
mTORC1
and
mTORC2,
the
downstream
effectors
of
PI3K/AKT
sig-
naling,
induce
EMT,
motility,
and
metastasis
of
CRC
via
RhoA
and
Rac1
signaling
[92].
Further,
cross-talk
of
the
Wnt
and
PI3K–AKT
signaling
pathways
at
GSK3␤
results
in
stabilization
and
accumu-
lation
of
␤-catenin
and
SNAI1
[93].
Also,
many
epidemiological
studies
have
been
demonstrated
that
a
high-fat
diet
(HFD)
is
cor-
related
with
a
high
prevalence
of
colon
cancer
[94].
A
recent
report
suggests
that
an
HFD
can
mediate
the
development
of
the
EMT
and
tumor
inflammation
through
up-regulation
of
COX-2
and
acti-
vation
of
MAPK/ERK
and
PI3K/AKT/mTOR
signaling
cascades
in
a
mouse
xenograft
model
of
CRC
[95].
2.3.
Non-coding
RNAs
in
EMT
Small
non-coding
RNAs
or
microRNAs
(miRNAs),
which
are
19–24
nucleotides
in
length,
control
gene
expression
posttranscrip-
tionally,
either
by
degrading
target
mRNAs
or
inhibiting
protein
translation.
Approximately
50%
of
human
miRNAs
are
located
at

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et
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Research
and
Practice
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(2015)
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561
Table
2
Molecular
factors
involved
in
EMT
and
CRC
progression:
non-coding
RNAs.
Molecules
Upstream
regulators
Target
genes
Clinical
significance
in
CRC
Refs.
miRNAs
involved
in
inhibiting
EMT
miR-200
family P53/TGF-␤/ZEB1/SNAI1/SIX1 ZEB1/ETS1/FLT1
Suppressor
of
poor
survival
and
poor
prognosis
[100–107]
miR-34
P53/SNAI1/ZEB1/IL-6
Fra-1/ZNF281/SNAI1
Suppressor
of
lymph
node
and
distant
CRC
metastasis
[108–111]
miR-15a/16-1
P53/AP4
AP4
Suppressor
of
distant
metastasis
and
poor
survival
[112]
miR-183-96-182
p21-ZEB1
SLUG/ZEB1
ND
[55]
miR-137
HMGA1
FMNL2
ND
[114]
miR-138
ND
TWIST2
Suppressor
of
lymph
node
metastasis,
distant
metastasis
and
poor
prognosis
[116]
miR-212
ND
MnSOD
Suppressor
of
a
more
aggressive
tumor
phenotype
and
poor
progress
[117]
miR-30b
ND
SIX1
Suppressor
of
liver
metastasis
and
poor
survival
[118]
miR-320a
ND
Rac1
Suppressor
of
metastasis
[119]
miR-101
HIF-1␣
␤-catenin
ND
[120]
miRNAs
involved
in
promoting
EMT
miR-21
TGF-␤/TNF-␣/AP1/ETS1 TIAM1/RECK
Integrin-␤4 Poor
prognosis [122–125]
miR-31
TGF-␤/TNF-␣
TIAM1/SATB2
Poor
prognosis
[122,126]
miR-9
PROX1
E-cadherin
ND
[45]
miR-130b
ND
PPAR␥
Distant
metastasis,
poor
survival
and
poor
prognosis
[127]
miR-29a
ND
KLF4
Metastasis
and
poor
prognosis
[128]
miR-103/107
ND
DAPK/KLF4
Lymph
node
and
distant
metastasis,
poor
survival
and
poor
prognosis
[129]
Abbreviations:
EMT,
epithelial–mesenchymal
transition;
CRC,
colorectal
cancer;
miR,
microRNA;
ND,
not
determined;
*hypothesis;
TGF-␤,
transforming
growth
factor-␤;
SIX1,
SIX
homeobox
1;
FLT1,
fms-related
tyrosine
kinase
1;
ZNF281,
finger
protein
281;
MMP,
matrix
metalloproteinase;
PBX3,
pre-B-cell
leukemia
homeobox
3;
HMGA1,
high-mobility
group
A1;
FMNL2,
formin-like
2;
MnSOD,
manganese
superoxide
dismutase;
Rac1,
ras-related
C3
botulinum
toxin
substrate
1;
HIF-1␣,
hypoxia-inducible
factor
1-␣;
TNF-␣,
tumor
necrosis
factor-␣;
TIAM1,
T-cell
lymphoma
invasion
and
metastasis
1;
AP1,
activator
protein
1;
RECK,
reversion-inducing-cysteine-rich
protein
with
kazal
motifs;
SATB2,
special
AT-rich
sequence-binding
protein
2;
PROX1,
prospero
homeobox
1;
PPAR␥,
peroxisome
proliferator-activated
receptor
␥;
KLF4,
kruppel-like
factor
4;
DAPK,
death-associated
protein
kinase.
chromosomal
breakpoints
and
are
susceptible
to
dysregulation
in
cancer
[96].
In
recent
years,
the
number
of
miRNAs
that
have
been
directly
or
indirectly
associated
with
the
EMT
have
become
as
extensive
as
the
list
of
EMT-associated
transcription
factors
(Table
2).
Some
research
has
focused
on
the
highly
conserved
miR-200
family
(miR-200a/b/c,
miR-141
and
miR-429),
the
downregulation
of
which
is
believed
to
be
the
essential
feature
of
EMT.
These
stud-
ies
have
demonstrated
that
ZEB1
and
ZEB2
are
targets
of
miR-200c
and
miR-200b,
respectively
[97–99].
Loss
of
the
tumor
suppressor
p53,
which
activates
miR-200c
through
direct
binding
to
miR-200c
promoter
sites,
leads
to
activating
the
EMT
program
through
the
decreased
expression
of
miR-200c
[100].
Moreover,
the
miR-200
family
and
miR-205
are
downstream
molecules
of
the
TGF-␤
path-
way
in
EMT.
By
contrast,
ectopic
expression
of
these
miRNAs
leads
to
MET
in
cells
that
have
previously
undergone
EMT
[101,102].
Most
intriguingly,
ZEB1
also
directly
suppresses
the
transcription
of
miR-141
and
miR-200c,
which
strongly
activate
epithelial
dif-
ferentiation
in
CRC
cells
[103].
Thus,
a
reciprocal
feedback
loop
between
the
miR-200
family
and
the
ZEB
family
of
transcrip-
tion
factors
tightly
maintains
the
equilibrium
between
EMT
and
MET.
Epigenetic
regulation
of
the
different
epithelium-related
miR-
NAs
has
recently
attracted
much
attention.
It
has
recently
been
reported
that
miR-200a/200b/429
and
miR-200c/141
transcripts
undergo
dynamic
epigenetic
regulation
linked
to
EMT
or
MET.
In
normal
colon
mucosa,
the
crypts
(epithelial)
and
stroma
(mes-
enchymal)
are
already
unmethylated
and
methylated
at
the
5-CpG
islands
of
both
miR-200
loci,
respectively;
and
CRC
cells
undergo
selective
miR-200
hypermethylation
of
the
epithelial
component
[104].
These
findings
indicate
that
epigenetic
silencing
plasticity
of
the
miR-200
family
mediates
the
shift
between
EMT
and
MET
phenotypes
and
contributes
to
cancer
progression
and
metastasis
formation
in
human
tumorgenesis.
In
addition
to
that,
many
recent
clinical
studies
have
highlighted
the
role
of
miR-200
members
in
the
pathogenesis
of
metastatic
CRC.
Analysis
of
the
expression
and
methylation
status
of
54
pairs
of
primary
CRCs
and
corre-
sponding
matched
liver
metastatic
tissue
specimens
reveals
that
miR-200
and
miR-141
show
higher
expression
in
liver
metastasis
tissues
than
the
primary
CRC,
and
this
is
significantly
associated
with
hypomethylation
of
the
promoter
region
of
these
miRNAs.
Conversely,
the
invasion
front
in
primary
CRC
tissues
displays
low
miR-200c
expression
[102].
Consistent
with
this,
miR-200
is
sup-
pressed
in
the
initial
stages
of
stroma
invasion
but
is
restored
at
metastatic
sites
[105],
and
it
is
also
decreased
in
metastatic
compared
to
non-metastatic
CRC
[106].
This
low
microRNA-200
expression
is
associated
with
a
bad
prognosis
in
CRC
patients
[107].
Together,
these
studies
demonstrate
a
convincing
clinical
role
for
the
miR-200
family
in
CRC
and
make
them
suitable
as
clinical
mark-
ers.
Apart
from
the
miR-200
family,
other
anti-metastatic
miRNAs
are
also
decreased
in
CRC.
MiR-34a,
a
transcriptional
target
of
p53,
is
able
to
decrease
the
expression
of
MMP1
and
MMP9
via
tar-
geting
to
Fra-1
[108].
However,
decreased
miR-34a
expression
is
associated
with
upregulation
of
the
c-Met,
SNAI1,
and
␤-catenin
protein
levels,
which
are
correlated
with
distant
metastasis
of
CRC
[109].
MiR-34
and
SNAI1
can
form
a
double-negative
feedback
loop
to
regulate
EMT
directly
[110]
or
promote
EMT
by
coherent
feed-forward
regulation
of
ZNF281,
which
contributes
to
metas-
tasis
formation
in
CRC
[50].
In
addition,
an
IL-6R/STAT3/miR-34a
feedback
loop
with
respect
to
the
EMT
contributes
to
CRC
metas-
tasis,
and
p53
can
disrupt
this
loop
by
inducing
miR-34a
[111].
Besides,
there
is
another
double-negative
feedback
loop
involv-
ing
p53-induced
miR-15a/16-1
and
AP4,
which
regulates
EMT
and
metastasis
in
CRC
[112].
As
such,
aberrant
regulation
of
p53
sig-
naling
plays
critical
roles
in
CRC
metastasis
by
triggering
the
EMT.
The
tumor
suppressor
p21
forms
a
p21–ZEB1
complex
to
inhibit
EMT
by
regulating
the
expression
of
the
miR-183-96-182
cluster,
the
targets
of
which
are
ZEB1
and
SLUG,
implying
the
existence
of
a
reciprocal
feedback
loop.
Though
p53
directly
regulates
p21,
p53
does
not
regulate
the
EMT
by
this
miRNA
cluster
[56].
In
conclusion,

562
H.
Cao
et
al.
/
Pathology
–
Research
and
Practice
211
(2015)
557–569
Fig.
3.
New
players:
feedback
loops
by
microRNAs
regulating
EMT
in
CRC.
ZEB1/2
and
the
miR-200
family
form
a
feedforward
loop
to
stabilize
epithelial
and
mes-
enchymal
states,
respectively.
The
p53-induced
miR-34
family
and
miR-15a/16-1
also
form
double-negative
feedback
loops
with
the
crucial
EMT
activator
SNAI1
and
AP4,
respectively.
Similarly,
SNAI1
and
miR-34
feed-forward
regulation
of
ZNF281
is
able
to
promote
an
EMT.
Besides,
the
p21-ZEB1
complex
inhibits
the
EMT
through
the
miR-183-96-182
cluster,
the
targets
of
which
are
ZEB1
and
SNAI1.
The
feed-
back
loop
of
IL-6/IL-6R/STAT3/miR-34a
with
respect
to
EMT
also
contributes
to
CRC
metastasis.
Therefore,
these
circuits
seem
to
represent
a
new
unifying
mechanism,
which
is
deregulated
in
cancer.
these
miRNA/TF
feedback-loop
circuits
represent
a
new
unifying
mechanism
regulating
the
EMT
process
(Fig.
3).
Let-7c,
one
of
the
tumor-suppressing
miRNAs,
also
functions
as
a
metastasis
suppres-
sor
in
CRC
by
directly
destabilizing
the
mRNAs
of
MMP11
and
PBX3
at
least.
Down-regulation
of
let-7c
in
primary
cancer
tissues
is
sig-
nificantly
associated
with
metastases,
advanced
TNM
stages,
and
poor
survival
of
CRC
patients
[113].
MiR-137,
an
HMGA1
target,
suppresses
metastasis
in
a
CRC
mouse
model
by
directly
target-
ing
FMNL2
[114],
which
is
involved
in
the
TGF-␤-induced
EMT
via
Smad3
effectors
or
in
cooperation
with
the
MAPK/MEK
signal-
ing
circuit
[115].
Further,
miR-138
[116],
miR-212
[117],
miR-30b
[118],
miR-320a
[119],
and
miR-101
[120]
also
play
pivotal
roles
in
regulating
of
the
EMT
and
CRC
metastasis.
Conversely,
many
pro-metastatic
miRNAs
also
play
important
regulatory
roles
in
the
EMT.
On
the
basis
of
a
large
dataset
of
CRC
miRNAs,
of
82
that
are
modulated
during
tumor
progression,
22
are
involved
in
the
EMT
[121].
Upregulation
of
the
EMT
activator
TGF-␤
elevates
the
expression
of
miR-21
and
miR-31.
Consistently,
overexpression
of
these
miRNAs
significantly
accelerates
the
effect
of
TGF-␤-induced
EMT
[122].
Recent
work
showed
that
the
histone
posttranslational
modifications
associated
with
an
active
transcrip-
tional
state
(H3K9-14ac,
H3K3me3,
and
H3K27ac)
and
with
an
inactive
transcriptional
state
(H3K27me3
and
H3K9me2)
of
miR-
21
are
required
for
its
overexpression,
and
the
combination
of
high
miR-21
with
low
integrin-␤4
and
PDCD4
plays
an
important
role
in
regulating
the
EMT
[123].
Besides,
high
expression
of
miR-21
might
predict
a
poor
prognosis
in
patients
with
CRC
[124,125].
These
results
highlight
a
vital
pro-metastatic
mechanism
mediated
by
miR-21
in
CRC
progression.
MiR-31
is
increased
in
human
primary
CRC
tissues
with
lymph
node
metastases
and
its
elevation
induces
the
EMT
by
targeting
SATB2,
a
gene
associated
with
CRC
metas-
tasis
[126].
Besides,
the
protein
PROX1
noted
above
can
bind
to
the
miR-9-2
promoter
and
trigger
its
expression
to
directly
inhibit
E-cadherin
expression
[45].
The
other
pro-metastatic
miRNAs,
including
miR-130b
[127],
miR-29a
[128],
and
miR-103/107
[129],
also
promote
tumor
development
by
triggering
the
EMT.
Additional
regulatory
mechanisms
dependent
on
non-coding
RNAs
control
the
expression
of
several
EMT-TFs.
For
example,
long
non-coding
RNAs
(lncRNAs),
a
new
class
larger
than
200
nt
generated
from
regions
downstream
of
the
SNAI1
and
SLUG
genes,
increases
the
expres-
sion
of
these
two
EMT
inducers
[130].
It
has
been
reported
that
HOTAIR
promotes
tumor
metastasis
through
EMT
triggering
and
stemness
maintenance
in
CRC
and
breast
cancer
[131].
Further-
more,
the
latest
results
that
MALAT1
and
lncRNA–ATB
activated
by
TGF-␤
promote
metastasis
through
inducing
the
EMT
in
blad-
der
cancer
[132]
and
Hepatocellular
carcinoma
[133]
pave
the
way
for
high
interest
in
this
new
regulatory
mechanism.
Clearly,
these
current
studies
provide
evidence
that
non-coding
RNAs
play
impor-
tant
and
complex
roles
in
the
regulation
of
epithelial
plasticity
and
can
be
potent
candidates
as
potential
targets
of
cancer
ther-
apy.
2.4.
Microenvironmental
regulation
of
EMT
The
mutual
and
interdependent
interactions
between
cancer
cells
and
their
microenvironment
are
important
determinants
of
cancer
progression
toward
metastasis.
The
tumor
environment
is
a
complex
network
of
inflammatory
and
immune
cells,
carcinoma-
associated
fibroblasts
(CAFs),
hypoxia,
soluble
factors,
signaling
molecules,
and
extracellular
matrix
components
(Fig.
2).
In
a
par-
allel
investigation
of
the
invasive
front
and
tumor
center
area
of
primary
CRC,
the
presence
of
EMT
markers
at
the
invasive
front
implies
that
the
tumor
microenvironment
may
induce
the
occur-
rence
of
EMT
in
cancer
cells
[30,77].
CAFs
actively
take
part
in
the
interactions
between
tumor
cells
and
other
cell
types
in
the
tumor
microenvironment
and
also
mod-
ulate
the
microenvironment
by
producing
MMPs,
MMP
inhibitors,
ECM
components,
growth
factors
and
cytokines
[134].
Co-culture
assays
of
primary
CAFs
with
different
colon
cancer
cells
imply
pro-migratory
effects
on
tumor
cells
[135],
and
collagen
type
XII
has
been
found
to
be
a
novel
candidate
marker
of
myofibroblasts
and/or
cancer
cells
undergoing
de-differentiation
at
the
invasive
front
of
CRC
[136].
A
recent
study
found
that
CAFs
show
elevated
expression
of
genes
related
to
hypoxia,
EMT,
and
TGF-␤
path-
way
activation.
Most
importantly,
a
putative
TGF-␤
target
gene,
IGFBP7,
a
novel
tumor
stroma
marker
of
CAFs
and
endothelial
cells,
is
also
induced
in
the
EMT-like
phenotype
of
CRC
cells
[137].
Conversely,
our
laboratory
first
found
that
IGFBP7
could
inhibit
EMT
and
tumor
metastasis
by
repressing
TGF-beta-mediated
EMT
through
the
Smad
signaling
cascade
in
CRC
cells
[138].
IGFBP7
may
have
different
roles
in
specific
microenvironment.
Besides,
the
expression
of
tissue
inhibitor
of
metalloproteinases
1
(TIMP-1)
by
CAFs
at
the
invasive
front
is
directly
related
to
recurrence-free
survival
and
overall
survival
[139].
The
inflammatory
component
is
an
essential
part
of
the
malig-
nant
microenvironment.
IL-1␤,
a
pleiotropic
pro-inflammatory
cytokine,
promotes
colon
tumor
invasion
by
activating
the
EMT
[140].
Further,
IL-8
(CXCL8)
and
CXCL20
are
reported
to
synergize
to
promote
CRC
progression
and
metastasis
by
collaborative
induction
of
the
EMT
via
PI3K/AKT-ERK1/2
crosstalk
[141].
In
a
colitis-
associated
premalignant
cancer
model,
macrophage-derived
IL-6
expression
is
increased
in
the
colon
[142].
Treatment
of
CRC
cells
with
IL-6
results
in
an
EMT
mediated
by
direct
repres-
sion
of
miR-34a
through
the
activation
of
oncogenic
STAT3,
and
SNAI1
is
an
important
downstream
effector
of
IL-6R/STAT3/miR-
34a
with
respect
to
the
EMT.
Intriguingly,
IL-6R,
which
mediates
IL-6-dependent
STAT3
activation,
is
a
direct
target
of
miR-34a.
This
active
feedback
loop
is
necessary
for
EMT-mediated
CRC
invasion
and
metastasis
and
is
associated
with
nodal
and
distant
metastasis
in
CRC
patients.
Ectopic
expression
of
p53
can
disrupt
this
IL-6R/STAT3/miR-34a
feedback
circuit
by
inducing
miR-34a
[111].
On
the
other
hand,
IL-6
also
contributes
to
down-regulation
of
the
expression
and
activity
of
p53
by
stimulating
ribosome
biogenesis.
This
p53
downregulation
induces
EMT-like
cellular

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563
phenotypic
changes,
such
as
a
reduction
of
E-cadherin
and
an
increase
of
SLUG
[143].
Our
laboratory
also
found
that
aberrant
IL-
6/STAT3/Fra-1
signaling
axis
lead
to
CRC
aggressiveness
through
EMT
induction
[144].
Besides,
the
pro-inflammatory
cytokine
TNF-␣
has
been
implicated
in
inducing
the
EMT
in
CRC
by
AKT/GSK-
3␤-mediated
stabilization
of
SNAI1,
and
this
finding
reinforces
the
connection
between
inflammation
and
the
EMT
[145].
In
addition
to
inducing
the
EMT
directly,
TNF-␣
can
accelerate
a
TGF-␤-induced
EMT
dramatically
by
increasing
IL-8
and
CXCR1
[146].
By
proteome
profiling
of
the
CAFs,
LTBP2,
CDH11,
OLFML3
and
FSTL1,
which
are
all
TGF-␤
signaling-related
proteins,
are
identified
as
novel
pro-
inflammatory
signatures
of
the
cancer
stroma
[147].
In
the
majority
of
CRC
samples,
nuclear-Smad3,
a
marker
of
TGF-␤
signaling,
stains
predominantly
the
stromal
compartment.
Secretion
of
IL-11
by
TGF-␤-stimulating
CAFs
triggers
GP130/STAT3
signaling
for
metas-
tasis
initiation
[2].
Among
the
EMT–TFs,
SNAI1
is
expressed
in
CAFs
and
contributes
to
the
pro-tumorigenic
effects
of
CAFS
on
CRC
cells.
The
target
cytokine
MCP-3
may
be
implicated
in
this
SNAI1-
dependent
paracrine
effect
[148].
Significantly,
TGF-␤
is
also
the
most
potent
inducer
of
SNAI1
transcription.
SNAI1
can
activate
the
expression
of
the
proinflammatory
mediator
IL-8
by
directly
binding
to
its
E3/E4
E-boxes
[149].
Thus
the
relationship
between
inflammation
and
the
EMT
seems
to
be
an
interactive
feature
in
cancer
metastasis.
Other
types
of
cells
in
the
tumor
environment
are
involved
in
metastasis.
By
quantitative
secretome
analysis
of
the
co-cultured
cellular
supernatants
from
epithelia
and
colon
tumor
cells,
the
putative
oncosuppressor
IGFBP6
has
been
screened
to
decrease
in
the
co-cultured
system
and
proves
to
be
dramatically
downregu-
lated
in
EMT
cells
[150].
Smad4,
which
is
activated
by
TGF-␤
and
frequently
lost
in
CRC,
can
bind
directly
to
the
CCL15
gene
promoter
to
negatively
regulate
its
expression.
Loss
of
Smad4
promotes
CCL15
expression
to
recruit
CCR1+myeloid
cells
in
humans
and
in
a
mouse
model.
CCR1+myeloid
cells
facilitate
liver
metastasis
by
producing
MMP9,
allowing
cancer
cells
to
invade
surrounding
stro-
mal
tissues
at
the
invasion
front
[151].
In
turn,
inactivation
of
CCR1
blocks
the
accumulation
of
immature
myeloid
cells
and
suppresses
liver
metastasis
[152].
Likewise,
mesenchymal
stem/stromal
cells
(MSCs),
which
are
multipotent
precursors
involved
in
chronic
inflammation,
can
trigger
the
EMT
in
CRC
cells
mainly
by
the
surface-bound
TGF-␤
newly
expressed
on
MSCs
upon
direct
co-
culture
with
tumor
cells.
Nevertheless,
exposure
to
their
released
soluble
pro-inflammatory
and
pro-angiogenic
factors,
including
IL-
6,
MCP1,
and
angiogenin,
is
not
sufficient
to
induce
an
EMT.
It
is
possible
that
some
MMPs
released
in
co-culture
supernatants,
in
particular
MMP-3,
contribute
to
the
cleavage
of
latent
TGF-␤
into
the
active
form
[153].
Platelets
are
also
contributors
to
metasta-
sis,
including
CRC
and
breast
cancer
[154].
A
recent
study
showed
that
platelet-derived
TGF-␤
and
direct
platelet–tumor
cell
inter-
actions
synergistically
induce
the
EMT
and
promote
metastasis
by
activating
the
TGF-␤
and
NF-␬B
pathways
[155].
Platelet-derived
growth
factor
receptor
B
(PDGFRB)
is
mainly
expressed
by
stromal
cells.
In
three
CRC
cohorts,
PDGFRB
is
expressed
in
primary
CRC
and
co-expressed
with
genes
involved
in
platelet
activation,
TGF-␤
sig-
naling,
and
the
EMT.
The
activation
of
platelets,
the
major
source
of
PDGF
and
TGF-␤,
causes
robust
PDGFRB
phosphorylation
on
tumor
cells
and
then
gives
rise
to
an
aggressive
phenotype
of
CRCs
with
mesenchymal
properties
[156].
Tumor-associated-endothelial
cells
(ECs)
also
play
a
critical
role
in
promoting
tumor
metastasis.
The
CRC-derived-fibronectin
extra
domain
A
(EDA)
secreted
by
ECs
induces
an
EMT
via
the
interaction
with
integrin␣9␤1
on
neigh-
boring
CRC
cells.
This
reveals
a
new
understanding
of
how
ECs
contribute
to
cancer
progression
[157].
From
a
therapeutic
stand-
point,
these
findings
support
the
idea
that,
in
addition
to
targeting
the
cancer
cell,
targeting
the
stromal
cells
or
stroma–tumor
cross-
talk
should
be
considered
as
a
potential
antimetastatic
approach.
3.
Malignant
phenotypes
of
EMT
in
colorectal
cancer
3.1.
EMT
and
tumor
budding
In
CRC,
tumor
cells
that
have
undergone
the
EMT
are
histo-
logically
represented
by
the
presence
of
tumor
buds
[158],
which
is
defined
as
an
isolated
single
cancer
cell
or
a
cluster
composed
of
fewer
than
five
undifferentiated
cancer
cells
scattered
in
the
stroma
at
the
invasive
front
[159].
The
frequency
of
tumor
budding
increases
with
more
advanced
TNM
stages
[160],
and
is
predictive
of
lymph
node
metastasis,
vascular
and
lymphatic
invasion
as
well
as
distant
metastasis
[161,162],
local
recurrence,
and
poor
disease-
specific
survival
time
[163,164].
It
has
also
has
been
classified
as
an
additional
prognostic
factor
by
the
UICC
[165].
The
first
step
in
a
tumor
bud’s
life
seems
to
be
its
detachment
from
the
main
tumor
body
by
loss
of
the
adhesion
molecule
E-
cadherin.
Indeed,
aggressive
tumor
buds
also
express
fibronectin
within
the
cytoplasm,
implying
a
more
mesenchymal
phenotype
underling
the
interaction
between
tumor
buds
and
the
surround-
ing
stroma
[166].
The
microenvironment
at
the
invasive
front
is
important
for
the
formation
of
tumor
buds
in
CRC.
It
has
been
reported
that
TGF-␤
is
an
important
cytokine
involved
in
the
cross-
talk
between
tumor
cells
and
stroma
cells
[2,59].
The
infiltration
of
CD10+myeloid
cells
at
the
invasion
front
interact
with
TGF-␤
and
enhance
the
tumor-budding
grade
[167].
Selective
and
tran-
sient
loss
of
the
basement
membrane
(BM)
at
the
invasive
front
is
correlated
with
increased
distant
metastasis
and
poor
patient
sur-
vival
in
CRC.
This
transient
loss
is
linked
to
an
EMT
in
tumor
cells;
ZEB1
is
the
crucial
transcriptional
repressor
of
BM
components
[29],
and
this
activation
of
ZEB1
may
be
due
to
aberrant
expression
of
SIX1
and
down-regulation
of
the
miR-200
family
[46,102,105].
Furthermore,
ZEB1
and
SNAI1-positive
cells
with
a
mesenchymal
morphology
at
the
invasive
front
show
strong
nuclear
␤-catenin
signals
(due
to
an
APC
mutation)
and
concomitant
loss
of
E-
cadherin
expression,
suggesting
that
these
tumor-budding
cells
have
undergone
an
EMT
[77,78].
In
addition,
recent
evidence
has
revealed
a
tight
association
between
C4.4A
and
tumor
budding,
in
part,
due
to
C4.4A
promoting
the
EMT
at
the
invasive
front
of
CRC
[168].
Tumor
buds
also
play
a
role
in
the
ECM
degradation
that
is
implicated
in
the
EMT
by
increasing
overexpression
of
matrix
metalloprotease
9
(MMP9),
and
cathepsin
B
[169],
urokinase
plasminogen-activator
receptor
(uPAR)
[170],
and
matrilysin
and
laminin5␭2
in
cases
with
high-grade
tumor-budding
[171].
Addi-
tional
studies
have
related
tumor
budding
to
increased
expression
of
the
putative
stem
cell
markers
CD44,
CD133,
and
ABCG5,
as
well
as
beta-III
tubulin,
CXCL12,
hMena,
and
cathepsin
B
[172].
3.2.
EMT
and
drug
resistance
Resistance
to
drug
therapy
is
a
serious
problem
for
clinicians
treating
a
variety
of
solid
cancers,
including
lung,
breast,
colorectal
and
pancreatic
cancers.
An
increasing
number
of
findings
suggest
that
tumors
undergoing
EMT
resist
conventional
drug
therapy.
Oxaliplatin-resistant
(OxR)
CRC
cells
exhibit
a
mesenchymal
mor-
phology
and
display
features
suggestive
of
EMT
(e.g.,
decreased
E-cadherin
and
increased
vimentin)
[173]
and
this
may
be
due
in
part
to
the
Fas
signaling
activated
by
oxaliplatin
[174].
Con-
versely,
studies
of
CRC
cells
have
shown
that
expression
of
the
EMT
inducers
SNAI1
[175]
or
TGF-␤
in
Smad4-deficient
cells
[68]
increase
the
resistance
to
5-fluorouracil.
The
adjuvant
calcitriol
can
enhance
radiation
sensitivity
in
CRC,
but
expression
of
the
EMT
mediator
SLUG
confers
resistance
to
this
effect
[176].
Con-
sistent
with
this,
C-terminal
tensin-like
(CTEN)
[177]
and
IL-1␤
[140],
EMT-related
inducers,
increase
drug
resistance
to
carbo-
platin
and
staurosporine,
respectively.
In
turn,
forced
expression
of

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miR-200c,
the
negative
regulator
of
the
EMT,
restores
chemother-
apeutic
sensitivity
[178].
In
clinical,
tumor
specimens
taken
from
CRC
patients
who
have
received
preoperative
chemotherapy
fol-
lowed
by
radical
surgery
display
phenotypic
changes
characteristic
of
and
molecular
changes
consistent
with
the
EMT.
Consistently,
loss
of
E-cadherin
promotes
drug
resistance
in
CRC
patients
[179]
and
the
recurrent
tumors
have
a
significantly
EMT-like
signature
[180].
SNAI1-expressing
cells
are
also
associated
with
resistance
to
dendritic-cell
immunotherapy
[181],
implying
that
inhibition
of
SNAI1-induced
EMT
simultaneously
suppresses
both
metastasis
and
immunosuppression
in
cancer
patients.
Besides
the
evidence
of
drug
resistance
to
chemotherapy
and
immunotherapy,
recent
studies
have
also
suggested
a
role
of
the
EMT
in
drug
resistance
to
targeted
therapies.
Pancreatic
and
CRC
cell
lines
insensitive
to
EGFR
inhibition
often
express
proteins
associated
with
an
EMT,
such
as
vimentin,
ZEB1
and
SNAI1.
In
turn,
epithelial
cells
show
an
aver-
age
7-fold
greater
sensitivity
than
mesenchymal-like
cells
[182],
implying
a
strong
correlation
between
EMT
activation
and
drug
resistance.
While
it
remains
unknown
how
the
EMT
program
impinges
on
drug
resistance,
several
possible
mechanism
may
be
in
play.
Most
importantly,
as
discussed
above,
the
EMT
program
gener-
ates
cells
with
CSC
properties.
CSCs
are
a
highly
self-renewing
population
and
are
resistant
to
conventional
anticancer
regimens.
This
is
mediated
by
a
combination
of
several
critical
features,
including
relative
dormancy,
efficient
DNA
repair,
high
expression
of
multidrug-resistance-type
membrane
transporters
and
protec-
tion
by
a
hypoxic
niche
environment
[183].
Rectal
CSCs
that
carry
both
CD44
and
CD54
surface
markers
exhibit
EMT
characteristics
and
are
resistant
to
both
5-fluorouracil/calcium
folinate/oxaliplatin
(FolFox)
and
cetuximab
treatment,
the
most
common
regimens
used
for
patients
with
advanced
or
metastatic
rectal
cancer
[184].
Further,
chemotherapy
increases
the
CD44high–CD24low cell
popu-
lation
and
these
cells
have
stem-cell
features
[185].
Accordingly,
these
findings
strongly
indicate
that
activation
of
the
EMT
con-
tributes
to
cancer
therapy
resistance
in
part
by
producing
cells
with
stem
cell-like
properties.
4.
Cancer
therapeutics
targeting
EMT
The
importance
of
the
EMT
in
the
acquisition
of
treatment
resistance
and
metastatic
properties
also
exposes
novel
therapeu-
tic
opportunities.
Preventing
or
reversing
EMT
processes
in
CRCs
may
be
a
promising
approach
to
reduce
metastases,
recurrence
and
resistance
to
neoadjuvant
therapy.
First,
it
is
generally
consid-
ered
that
small-molecule
compounds
that
restore
the
expression
of
E-cadherin
are
able
to
suppress
tumor
malignancy.
One
research
group
developed
a
fluorescence
screening
system
based
on
E-
cadherin
expression
and
found
that
methotrexate
(MTX)
and
two
compounds
strongly
induce
E-cadherin.
Although
MTX
is
not
used
in
CRC
therapy,
extensive
clinical
trials
might
improve
the
out-
comes.
The
other
two
leading
drug
candidates
have
high
potential
as
novel
cancer
drugs
aimed
at
preventing
metastasis
[186].
Fur-
ther
work
should
be
performed
to
elucidate
their
mechanisms
of
action
and
to
optimize
them
as
new
therapeutic
agents.
Second,
the
inducers
of
the
full
EMT
program
are
transcription
factors,
and
are
thus
very
difficult
to
target.
RNA
interference
provides
some
hope
in
terms
of
specificity,
but
further
development
is
needed
to
increase
the
stability
of
these
reagents
and
the
efficiency
in
cell
targeting
and
intracellular
delivery.
In
addition,
an
alterna-
tive
approach
to
drug
therapy
that
targets
transcription
factors
driving
the
metastatic
process
is
T-cell-mediated
immunotherapy.
Brachyury,
a
member
of
the
T-box
transcription-factor
family,
is
a
driver
of
the
EMT
in
a
range
of
human
cancers
including
col-
orectal
carcinoma
as
noted
above
[37,187,188].
Brachyury-specific
human
T-cells
that
are
capable
of
lysing
Brachyury-positive
tumor
cells
have
been
generated
in
vitro
[189].
A
phase
I
clinical
trial
of
a
recombinant
yeast-Brachyury
vector-based
vaccine
is
cur-
rently
ongoing
in
patients
with
advanced
tumors.
To
date,
this
is
the
first
vaccine
platform
designed
to
target
a
driver
of
the
EMT
in
tumors
and
has
successfully
reached
the
clinical
stage
[190].
Consistent
with
this,
a
recombinant
yeast-TWIST1
vaccine
also
has
anti-tumor
effects
that
depend
on
TWIST1-specific
CD8+
T-cells
[191].
Together,
these
studies
provide
the
rationale
for
vaccine-induced
T-cell-mediated
therapy
with
transcription
fac-
tors
involved
in
driving
the
metastatic
process
without
toxic
effects.
This
therapy
is
likely
to
be
applied
to
several
types
of
cancer
including
CRC
in
the
near
future.
Besides,
miRNAs
targeted
to
mRNAs
encoding
stem-cell
signaling
components
or
EMT
regula-
tors,
are
also
potent
drug
targets.
For
example,
it
has
been
reported
that
electrogene
therapy
with
miRNA-KRAS
may
be
a
therapeu-
tic
strategy
for
the
treatment
of
CRCs
harboring
KRAS
mutation
[192].
A
strategy
currently
under
way
is
to
target
the
membrane
recep-
tors
that
transmit
the
extracellular
signals
that
activate
the
EMT
program.
For
instance,
inhibitors
of
ALK5,
MEK,
and
SRC
could
inter-
fere
with
the
EMT
in
response
to
EGF,
HGF,
and
IGF-1
[193,194].
Celecoxib,
a
selective
cyclooxygenase-2
(COX2)
inhibitor,
has
the
potential
to
negatively
affect
the
induction
of
EMT
by
hypoxia
and/or
EGF
in
CRC
[195].
Furthermore,
rapamycin
and
17-AGG
have
been
identified
as
inhibitors
of
a
TGF-␤-induced
EMT
by
modifying
the
TGF-␤
pathway
[196].
P17
and
P144,
TGF-␤-blocking
pep-
tides,
reverse
the
TGF-␤-induced
EMT
and
CSC
phenotypes
[197],
suggesting
that
they
are
potential
therapeutic
options
for
the
treat-
ment
of
metastatic
CRC.
Besides,
salinomycin
has
also
been
shown
to
selectively
kill
E-cadherin-null
breast
epithelial
cells
compared
with
E-cadherin-positive
cells
[198],
although
its
molecular
mech-
anism
in
the
EMT
is
unknown.
Intriguingly,
John
Giannios
et
al.
used
immunochemogene
treatment
composed
of
a
stealth
nanoparticle
formulation,
consisting
of
clamp
PNA
against
the
mRNA
of
FOXC2,
anti-CD44
chimeric
MAb,
and
vinorelbine,
in
an
attempt
to
erad-
icate
metastatic
CRC
cells
and
inhibit
metastasis
by
blocking
the
EMT
[199].
Furthermore,
CTCs
have
the
promise
of
serving
as
liq-
uid
biopsies
for
tumors
with
potential
for
monitoring
the
efficacy
of
treatment
[200].
5.
Conclusions
and
future
perspectives
The
current
data
suggest
that
an
important
role
of
the
EMT
in
the
process
of
cancer
metastasis.
The
EMT
program
is
complex
and
con-
trolled
by
many
transcriptional
regulators
and
non-coding
RNAs.
The
EMT
process
is
also
associated
with
malignant
behaviors
in
CRC,
including
tumor
budding,
circulating
tumor
cells
and
drug
resis-
tance.
In
comparison
with
other
tumor
types
such
as
breast
cancer,
experimental
data
on
the
role
of
the
EMT
in
CRC
metastases
are
still
limited.
By
far,
the
relationship
between
the
tumor
microenvi-
ronment
and
the
EMT
is
not
yet
mature
and
need
more
basic
and
clinical
experiments
for
clarification.
As
for
non-coding
miRNAs,
current
studies
have
identified
some
lncRNAs
involved
in
the
EMT
program
in
different
cancer
types
and
these
lncRNAs
may
become
novel
therapeutic
targets.
However,
there
is
a
little
research
on
the
lncRNAs
in
CRC
and
more
studies
are
required
to
elucidate
their
functions.
Finally,
the
latest
report
in
a
mouse
model
of
pan-
creatic
cancer
has
revealed
that
pancreatic
epithelial
cells
invade
and
enter
the
bloodstream
at
a
very
early
premalignant
stage
such
as
the
pancreatitis
period,
and
the
induction
of
inflammation
promotes
cancer
progression
in
part
by
facilitating
the
EMT
and
increasing
cell
dissemination
[201].
These
results
provide
the
insight
that
CRC
progression
may
also
have
similar
phenomena
and
more
experimental
and
clinical
experiments
are
required
to

H.
Cao
et
al.
/
Pathology
–
Research
and
Practice
211
(2015)
557–569
565
clarify
the
exact
mechanism.
More
importantly,
these
studies
have
implications
for
the
management
of
individuals
at
high
risk
of
CRC,
including
patients
with
ulcerative
colitis
or
kindreds
with
inherited
CRC.
Acknowledgements
This
work
was
supported
by
grants
from
the
Natural
Science
Foundation
of
Zhejiang
Province
(LY12H16017),
Major
Program
of
the
National
Natural
Science
Foundation
of
China
(Nos.
81090420,
81090421),
111
Project
(No.
B13026)
and
Grant
2012C13014-3
from
Department
of
Science
and
Technology
of
Zhejiang
Province,
PRC.
We
thank
Dr
Iain
Charles
Bruce
(Department
of
Physiology,
School
of
Medicine,
Zhejiang
University)
for
linguistic
review
of
the
manuscript.
References
[1]
J.
Ferlay,
H.R.
Shin,
F.
Bray,
D.
Forman,
C.
Mathers,
D.M.
Parkin,
Estimates
of
worldwide
burden
of
cancer
in
2008:
GLOBOCAN
2008,
Int.
J.
Cancer
127
(2010)
2893–2917.
[2]
A.
Calon,
E.
Espinet,
S.
Palomo-Ponce,
D.V.
Tauriello,
M.
Iglesias,
M.V.
Cespedes,
et
al.,
Dependency
of
colorectal
cancer
on
a
TGF-beta-driven
program
in
stromal
cells
for
metastasis
initiation,
Cancer
Cell
22
(2012)
571–584.
[3]
H.
Brenner,
M.
Kloor,
C.P.
Pox,
Colorectal
cancer,
Lancet
(2013).
[4]
S.
Manfredi,
C.
Lepage,
C.
Hatem,
O.
Coatmeur,
J.
Faivre,
A.M.
Bouvier,
Epidemiology
and
management
of
liver
metastases
from
colorectal
cancer,
Ann.
Surg.
244
(2006)
254–259.
[5]
I.J.
Fidler,
The
pathogenesis
of
cancer
metastasis:
the
‘seed
and
soil’
hypothesis
revisited,
Nat.
Rev.
Cancer
3
(2003)
453–458.
[6]
B.
Boyer,
J.P.
Thiery,
Epithelium–mesenchyme
interconversion
as
example
of
epithelial
plasticity,
APMIS
101
(1993)
257–268.
[7]
J.P.
Thiery,
H.
Acloque,
R.Y.
Huang,
M.A.
Nieto,
Epithelial–mesenchymal
transitions
in
development
and
disease,
Cell
139
(2009)
871–890.
[8]
D.J.
Burgess,
Breast
cancer:
circulating
and
dynamic
EMT,
Nat.
Rev.
Cancer
13
(2013)
148.
[9]
P.
Mak,
I.
Leav,
B.
Pursell,
D.
Bae,
X.
Yang,
C.A.
Taglienti,
et
al.,
ERbeta
impedes
prostate
cancer
EMT
by
destabilizing
HIF-1alpha
and
inhibiting
VEGF-mediated
snail
nuclear
localization:
implications
for
Gleason
grading,
Cancer
Cell
17
(2010)
319–332.
[10]
W.
Zhu,
M.Y.
Cai,
Z.T.
Tong,
S.S.
Dong,
S.J.
Mai,
Y.J.
Liao,
et
al.,
Overexpression
of
EIF5A2
promotes
colorectal
carcinoma
cell
aggressiveness
by
upregulating
MTA1
through
C-myc
to
induce
epithelial–mesenchymal
transition,
Gut
61
(2012)
562–575.
[11]
C.
Pena,
J.M.
Garcia,
J.
Silva,
V.
Garcia,
R.
Rodriguez,
I.
Alonso,
et
al.,
E-cadherin
and
vitamin
D
receptor
regulation
by
SNAIL
and
ZEB1
in
colon
cancer:
clinicopathological
correlations,
Hum.
Mol.
Genet.
14
(2005)
3361–3370.
[12]
X.
He,
Z.
Chen,
M.
Jia,
X.
Zhao,
Downregulated
E-cadherin
expression
indicates
worse
prognosis
in
Asian
patients
with
colorectal
cancer:
evidence
from
meta-analysis,
PLOS
ONE
8
(2013)
e70858.
[13]
E.D.
Hay,
An
overview
of
epithelio-mesenchymal
transformation,
Acta
Anat.
(Basel)
154
(1995)
8–20.
[14]
A.
Vincent-Salomon,
J.P.
Thiery,
Host
microenvironment
in
breast
cancer
development:
epithelial–mesenchymal
transition
in
breast
cancer
development,
Breast
Cancer
Res.
5
(2003)
101–106.
[15]
Y.
Toiyama,
H.
Yasuda,
S.
Saigusa,
K.
Tanaka,
Y.
Inoue,
A.
Goel,
et
al.,
Increased
expression
of
Slug
and
Vimentin
as
novel
predictive
biomarkers
for
lymph
node
metastasis
and
poor
prognosis
in
colorectal
cancer,
Carcinogenesis
34
(2013)
2548–2557.
[16]
E.
Batlle,
E.
Sancho,
C.
Franci,
D.
Dominguez,
M.
Monfar,
J.
Baulida,
et
al.,
The
transcription
factor
snail
is
a
repressor
of
E-cadherin
gene
expression
in
epithelial
tumour
cells,
Nat.
Cell
Biol.
2
(2000)
84–89.
[17]
A.
Cano,
M.A.
Perez-Moreno,
I.
Rodrigo,
A.
Locascio,
M.J.
Blanco,
M.G.
Del
Barrio,
et
al.,
The
transcription
factor
snail
controls
epithelial–mesenchymal
transitions
by
repressing
E-cadherin
expression,
Nat.
Cell
Biol.
2
(2000)
76–83.
[18]
K.M.
Hajra,
D.Y.
Chen,
E.R.
Fearon,
The
SLUG
zinc-finger
protein
represses
E-cadherin
in
breast
cancer,
Cancer
Res.
62
(2002)
1613–1618.
[19]
J.
Comijn,
G.
Berx,
P.
Vermassen,
K.
Verschueren,
L.
Van
Grunsven,
E.
Bruyneel,
et
al.,
The
two-handed
E
box
binding
zinc
finger
protein
SIP1
downregulates
E-cadherin
and
induces
invasion,
Mol.
Cell
7
(2001)
1267–1278.
[20]
A.
Eger,
K.
Aigner,
S.
Sonderegger,
B.
Dampier,
S.
Oehler,
M.
Schreiber,
et
al.,
DeltaEF1
is
a
transcriptional
repressor
of
E-cadherin
and
regulates
epithelial
plasticity
in
breast
cancer
cells,
Oncogene
24
(2005)
2375–2385.
[21]
J.
Yang,
S.A.
Mani,
J.L.
Donaher,
S.
Ramaswamy,
R.A.
Itzykson,
C.
Come,
et
al.,
Twist,
a
master
regulator
of
morphogenesis,
plays
an
essential
role
in
tumor
metastasis,
Cell
117
(2004)
927–939.
[22]
X.
Fang,
Y.
Cai,
J.
Liu,
Z.
Wang,
Q.
Wu,
Z.
Zhang,
et
al.,
Twist2
contributes
to
breast
cancer
progression
by
promoting
an
epithelial–mesenchymal
transition
and
cancer
stem-like
cell
self-renewal,
Oncogene
30
(2011)
4707–4720.
[23]
M.A.
Perez-Moreno,
A.
Locascio,
I.
Rodrigo,
G.
Dhondt,
F.
Portillo,
M.A.
Nieto,
et
al.,
A
new
role
for
E12/E47
in
the
repression
of
E-cadherin
expression
and
epithelial–mesenchymal
transitions,
J.
Biol.
Chem.
276
(2001)
27424–27431.
[24]
F.
Kroepil,
G.
Fluegen,
D.
Vallbohmer,
S.E.
Baldus,
L.
Dizdar,
A.M.
Raffel,
et
al.,
Snail1
expression
in
colorectal
cancer
and
its
correlation
with
clinical
and
pathological
parameters,
BMC
Cancer
13
(2013)
145.
[25]
M.
Shioiri,
T.
Shida,
K.
Koda,
K.
Oda,
K.
Seike,
M.
Nishimura,
et
al.,
Slug
expression
is
an
independent
prognostic
parameter
for
poor
survival
in
colorectal
carcinoma
patients,
Br.
J.
Cancer
94
(2006)
1816–1822.
[26]
M.J.
Larriba,
E.
Martin-Villar,
J.M.
Garcia,
F.
Pereira,
C.
Pena,
A.G.
De
Herreros,
et
al.,
Snail2
cooperates
with
Snail1
in
the
repression
of
vitamin
D
receptor
in
colon
cancer,
Carcinogenesis
30
(2009)
1459–1468.
[27]
I.
Gomez,
C.
Pena,
M.
Herrera,
C.
Munoz,
M.J.
Larriba,
V.
Garcia,
et
al.,
TWIST1
is
expressed
in
colorectal
carcinomas
and
predicts
patient
survival,
PLoS
ONE
6
(2011)
e18023.
[28]
A.B.
Singh,
A.
Sharma,
J.J.
Smith,
M.
Krishnan,
X.
Chen,
S.
Eschrich,
et
al.,
Claudin-1
up-regulates
the
repressor
ZEB-1
to
inhibit
E-cadherin
expression
in
colon
cancer
cells,
Gastroenterology
141
(2011)
2140–2153.
[29]
S.
Spaderna,
O.
Schmalhofer,
F.
Hlubek,
G.
Berx,
A.
Eger,
S.
Merkel,
et
al.,
A
transient,
EMT-linked
loss
of
basement
membranes
indicates
metastasis
and
poor
survival
in
colorectal
cancer,
Gastroenterology
131
(2006)
830–840.
[30]
C.
Kahlert,
S.
Lahes,
P.
Radhakrishnan,
S.
Dutta,
C.
Mogler,
E.
Herpel,
et
al.,
Overexpression
of
ZEB2
at
the
invasion
front
of
colorectal
cancer
is
an
independent
prognostic
marker
and
regulates
tumor
invasion
in
vitro,
Clin.
Cancer
Res.
17
(2011)
7654–7663.
[31]
E.
Sanchez-Tillo,
O.
De
Barrios,
L.
Siles,
P.G.
Amendola,
D.S.
Darling,
M.
Cuatrecasas,
et
al.,
ZEB1
Promotes
invasiveness
of
colorectal
carcinoma
cells
through
the
opposing
regulation
of
uPA
and
PAI-1,
Clin.
Cancer
Res.
19
(2013)
1071–1082.
[32]
K.M.
Welch-Reardon,
S.M.
Ehsan,
K.
Wang,
N.
Wu,
A.C.
Newman,
M.
Romero-Lopez,
et
al.,
Angiogenic
sprouting
is
regulated
by
endothelial
cell
expression
of
Slug,
J.
Cell
Sci.
127
(2014)
2017–2028.
[33]
G.
Celesti,
G.
Di
Caro,
P.
Bianchi,
F.
Grizzi,
G.
Basso,
F.
Marchesi,
et
al.,
Presence
of
Twist1-positive
neoplastic
cells
in
the
stroma
of
chromosome-unstable
colorectal
tumors,
Gastroenterology
145
(2013)
647–657,
e615.
[34]
F.
Valdes-Mora,
T.
Gomez
Del
Pulgar,
E.
Bandres,
P.
Cejas,
A.
Ramirez
De
Molina,
R.
Perez-Palacios,
et
al.,
TWIST1
overexpression
is
associated
with
nodal
invasion
and
male
sex
in
primary
colorectal
cancer,
Ann.
Surg.
Oncol.
16
(2009)
78–87.
[35]
T.
Okada,
Y.
Suehiro,
K.
Ueno,
S.
Mitomori,
S.
Kaneko,
M.
Nishioka,
et
al.,
TWIST1
hypermethylation
is
observed
frequently
in
colorectal
tumors
and
its
overexpression
is
associated
with
unfavorable
outcomes
in
patients
with
colorectal
cancer,
Genes
Chromosomes
Cancer
49
(2010)
452–462.
[36]
N.
Kilic,
S.
Feldhaus,
E.
Kilic,
P.
Tennstedt,
D.
Wicklein,
R.
Wasielewski,
et
al.,
Brachyury
expression
predicts
poor
prognosis
at
early
stages
of
colorectal
cancer,
Eur.
J.
Cancer
47
(2011)
1080–1085.
[37]
D.
Sarkar,
B.
Shields,
M.L.
Davies,
J.
Muller,
J.A.
Wakeman,
BRACHYURY
confers
cancer
stem
cell
characteristics
on
colorectal
cancer
cells,
Int.
J.
Cancer
130
(2012)
328–337.
[38]
R.
Jackstadt,
S.
Roh,
J.
Neumann,
P.
Jung,
R.
Hoffmann,
D.
Horst,
et
al.,
AP4
is
a
mediator
of
epithelial–mesenchymal
transition
and
metastasis
in
colorectal
cancer,
J.
Exp.
Med.
210
(2013)
1331–1350.
[39]
S.A.
Mani,
J.
Yang,
M.
Brooks,
G.
Schwaninger,
A.
Zhou,
N.
Miura,
et
al.,
Mesenchyme
Forkhead
1
(FOXC2)
plays
a
key
role
in
metastasis
and
is
associated
with
aggressive
basal-like
breast
cancers,
Proc.
Natl.
Acad.
Sci.
U.
S.
A.
104
(2007)
10069–10074.
[40]
T.
Watanabe,
T.
Kobunai,
Y.
Yamamoto,
K.
Matsuda,
S.
Ishihara,
K.
Nozawa,
et
al.,
Gene
expression
of
mesenchyme
forkhead
1
(FOXC2)
significantly
correlates
with
the
degree
of
lymph
node
metastasis
in
colorectal
cancer,
Int.
Surg.
96
(2011)
207–216.
[41]
E.
Sanchez-Tillo,
O.
De
Barrios,
L.
Siles,
M.
Cuatrecasas,
A.
Castells,
A.
Postigo,
Beta-catenin/TCF4
complex
induces
the
epithelial-to-mesenchymal
transition
(EMT)-activator
ZEB1
to
regulate
tumor
invasiveness,
Proc.
Natl.
Acad.
Sci.
U.
S.
A.
108
(2011)
19204–19209.
[42]
X.
Han,
X.
Fang,
X.
Lou,
D.
Hua,
W.
Ding,
G.
Foltz,
et
al.,
Silencing
SOX2
induced
mesenchymal–epithelial
transition
and
its
expression
predicts
liver
and
lymph
node
metastasis
of
CRC
patients,
PLoS
ONE
7
(2012)
e41335.
[43]
X.
Dai,
J.
Ge,
X.
Wang,
X.
Qian,
C.
Zhang,
X.
Li,
OCT4
regulates
epithelial–mesenchymal
transition
and
its
knockdown
inhibits
colorectal
cancer
cell
migration
and
invasion,
Oncol.
Rep.
29
(2013)
155–160.
[44]
H.M.
Meng,
P.
Zheng,
X.Y.
Wang,
C.
Liu,
H.M.
Sui,
S.J.
Wu,
et
al.,
Over-expression
of
Nanog
predicts
tumor
progression
and
poor
prognosis
in
colorectal
cancer,
Cancer
Biol.
Ther.
9
(2010)
295–302.
[45]
M.H.
Lu,
C.C.
Huang,
M.R.
Pan,
H.H.
Chen,
W.C.
Hung,
Prospero
homeobox
1
promotes
epithelial–mesenchymal
transition
in
colon
cancer
cells
by
inhibiting
E-cadherin
via
miR-9,
Clin.
Cancer
Res.
18
(2012)
6416–6425.
[46]
H.
Ono,
I.
Imoto,
K.
Kozaki,
H.
Tsuda,
T.
Matsui,
Y.
Kurasawa,
et
al.,
SIX1
promotes
epithelial–mesenchymal
transition
in
colorectal
cancer
through
ZEB1
activation,
Oncogene
31
(2012)
4923–4934.

566
H.
Cao
et
al.
/
Pathology
–
Research
and
Practice
211
(2015)
557–569
[47]
Y.
Takahashi,
G.
Sawada,
J.
Kurashige,
R.
Uchi,
T.
Matsumura,
H.
Ueo,
et
al.,
Paired
related
homoeobox
1,
a
new
EMT
inducer,
is
involved
in
metastasis
and
poor
prognosis
in
colorectal
cancer,
Br.
J.
Cancer
109
(2013)
307–311.
[48]
A.
Belton,
A.
Gabrovsky,
Y.K.
Bae,
R.
Reeves,
C.
Iacobuzio-Donahue,
D.L.
Huso,
et
al.,
HMGA1
induces
intestinal
polyposis
in
transgenic
mice
and
drives
tumor
progression
and
stem
cell
properties
in
colon
cancer
cells,
PLoS
ONE
7
(2012)
e30034.
[49]
J.
Diesch,
E.
Sanij,
O.
Gilan,
C.
Love,
H.
Tran,
N.I.
Fleming,
et
al.,
Widespread
FRA1-dependent
control
of
mesenchymal
transdifferentiation
programs
in
colorectal
cancer
cells,
PLoS
ONE
9
(2014)
e88950.
[50]
S.
Hahn,
R.
Jackstadt,
H.
Siemens,
S.
Hunten,
H.
Hermeking,
SNAIL
and
miR-34a
feed-forward
regulation
of
ZNF281/ZBP99
promotes
epithelial–mesenchymal
transition,
EMBO
J.
32
(2013)
3079–3095.
[51]
W.
Zhang,
B.
Jiang,
Z.
Guo,
C.
Sardet,
B.
Zou,
C.S.
Lam,
et
al.,
Four-and-a-half
LIM
protein
2
promotes
invasive
potential
and
epithelial–mesenchymal
transition
in
colon
cancer,
Carcinogenesis
31
(2010)
1220–1229.
[52]
W.
Zhang,
J.
Wang,
B.
Zou,
C.
Sardet,
J.
Li,
C.S.
Lam,
et
al.,
Four
and
a
half
LIM
protein
2
(FHL2)
negatively
regulates
the
transcription
of
E-cadherin
through
interaction
with
Snail1,
Eur.
J.
Cancer
47
(2011)
121–130.
[53]
E.H.
Nam,
Y.
Lee,
Y.K.
Park,
J.W.
Lee,
S.
Kim,
ZEB2
upregulates
integrin
alpha5
expression
through
cooperation
with
Sp1
to
induce
invasion
during
epithelial–mesenchymal
transition
of
human
cancer
cells,
Carcinogenesis
33
(2012)
563–571.
[54]
E.H.
Nam,
Y.
Lee,
X.F.
Zhao,
Y.K.
Park,
J.W.
Lee,
S.
Kim,
ZEB2-Sp1
cooperation
induces
invasion
by
upregulating
cadherin-11
and
integrin
alpha5
expression,
Carcinogenesis
35
(2014)
302–314.
[55]
E.
Sanchez-Tillo,
A.
Lazaro,
R.
Torrent,
M.
Cuatrecasas,
E.C.
Vaquero,
A.
Castells,
et
al.,
ZEB1
represses
E-cadherin
and
induces
an
EMT
by
recruiting
the
SWI/SNF
chromatin-remodeling
protein
BRG1,
Oncogene
29
(2010)
3490–3500.
[56]
X.L.
Li,
T.
Hara,
Y.
Choi,
M.
Subramanian,
P.
Francis,
S.
Bilke,
et
al.,
A
p21–ZEB1
complex
inhibits
epithelial–mesenchymal
transition
through
the
microRNA
183-96-182
cluster,
Mol.
Cell.
Biol.
34
(2014)
533–550.
[57]
K.
Polyak,
R.A.
Weinberg,
Transitions
between
epithelial
and
mesenchymal
states:
acquisition
of
malignant
and
stem
cell
traits,
Nat.
Rev.
Cancer
9
(2009)
265–273.
[58]
K.
Matsuzaki,
T.
Seki,
K.
Okazaki,
TGF-beta
during
human
colorectal
carcinogenesis:
the
shift
from
epithelial
to
mesenchymal
signaling,
Inflammopharmacology
14
(2006)
198–203.
[59]
P.
Lampropoulos,
A.
Zizi-Sermpetzoglou,
S.
Rizos,
A.
Kostakis,
N.
Nikiteas,
A.G.
Papavassiliou,
TGF-beta
signalling
in
colon
carcinogenesis,
Cancer
Lett.
314
(2012)
1–7.
[60]
J.
Massague,
How
cells
read
TGF-beta
signals,
Nat.
Rev.
Mol.
Cell
Biol.
1
(2000)
169–178.
[61]
Y.
Shi,
J.
Massague,
Mechanisms
of
TGF-beta
signaling
from
cell
membrane
to
the
nucleus,
Cell
113
(2003)
685–700.
[62]
K.A.
Brown,
J.A.
Pietenpol,
H.L.
Moses,
A
tale
of
two
proteins:
differential
roles
and
regulation
of
Smad2
and
Smad3
in
TGF-beta
signaling,
J.
Cell.
Biochem.
101
(2007)
9–33.
[63]
S.
Markowitz,
J.
Wang,
L.
Myeroff,
R.
Parsons,
L.
Sun,
J.
Lutterbaugh,
et
al.,
Inactivation
of
the
type
II
TGF-beta
receptor
in
colon
cancer
cells
with
microsatellite
instability,
Science
268
(1995)
1336–1338.
[64]
S.D.
Markowitz,
M.M.
Bertagnolli,
Molecular
origins
of
cancer:
molecular
basis
of
colorectal
cancer,
N.
Engl.
J.
Med.
361
(2009)
2449–2460.
[65]
A.
Reinacher-Schick,
S.E.
Baldus,
B.
Romdhana,
S.
Landsberg,
M.
Zapatka,
S.P.
Monig,
et
al.,
Loss
of
Smad4
correlates
with
loss
of
the
invasion
suppressor
E-cadherin
in
advanced
colorectal
carcinomas,
J.
Pathol.
202
(2004)
412–420.
[66]
T.J.
Freeman,
J.J.
Smith,
X.
Chen,
M.K.
Washington,
J.T.
Roland,
A.L.
Means,
et
al.,
Smad4-mediated
signaling
inhibits
intestinal
neoplasia
by
inhibiting
expression
of
beta-catenin,
Gastroenterology
142
(2012)
562–571,
e562.
[67]
B.
Zhang,
S.K.
Halder,
N.D.
Kashikar,
Y.J.
Cho,
A.
Datta,
D.L.
Gorden,
et
al.,
Antimetastatic
role
of
Smad4
signaling
in
colorectal
cancer,
Gastroenterology
138
(2010)
969–980,
e961–e963.
[68]
P.
Papageorgis,
K.
Cheng,
S.
Ozturk,
Y.
Gong,
A.W.
Lambert,
H.M.
Abdolmaleky,
et
al.,
Smad4
inactivation
promotes
malignancy
and
drug
resistance
of
colon
cancer,
Cancer
Res.
71
(2011)
998–1008.
[69]
M.
Pohl,
Y.
Radacz,
N.
Pawlik,
A.
Schoeneck,
S.E.
Baldus,
J.
Munding,
et
al.,
SMAD4
mediates
mesenchymal–epithelial
reversion
in
SW480
colon
carcinoma
cells,
Anticancer
Res.
30
(2010)
2603–2613.
[70]
S.
Zhao,
K.
Venkatasubbarao,
J.W.
Lazor,
J.
Sperry,
C.
Jin,
L.
Cao,
et
al.,
Inhibition
of
STAT3
Tyr705
phosphorylation
by
Smad4
suppresses
transforming
growth
factor
beta-mediated
invasion
and
metastasis
in
pancreatic
cancer
cells,
Cancer
Res.
68
(2008)
4221–4228.
[71]
H.
Xiong,
J.
Hong,
W.
Du,
Y.W.
Lin,
L.L.
Ren,
Y.C.
Wang,
et
al.,
Roles
of
STAT3
and
ZEB1
proteins
in
E-cadherin
down-regulation
and
human
colorectal
cancer
epithelial–mesenchymal
transition,
J.
Biol.
Chem.
287
(2012)
5819–5832.
[72]
L.
Losi,
H.
Bouzourene,
J.
Benhattar,
Loss
of
Smad4
expression
predicts
liver
metastasis
in
human
colorectal
cancer,
Oncol.
Rep.
17
(2007)
1095–1099.
[73]
P.W.
Voorneveld,
L.L.
Kodach,
R.J.
Jacobs,
N.
Liv,
A.C.
Zonnevylle,
J.P.
Hoogenboom,
et
al.,
Loss
of
SMAD4
alters
BMP
signaling
to
promote
colorectal
cancer
cell
metastasis
via
activation
of
Rho
and
ROCK,
Gastroenterology
147
(2014)
196–208,
e113.
[74]
M.
Hirakawa,
R.
Takimoto,
F.
Tamura,
M.
Yoshida,
M.
Ono,
K.
Murase,
et
al.,
Fucosylated
TGF-beta
receptors
transduces
a
signal
for
epithelial–mesenchymal
transition
in
colorectal
cancer
cells,
Br.
J.
Cancer
110
(2014)
156–163.
[75]
E.
Vincan,
N.
Barker,
The
upstream
components
of
the
Wnt
signalling
pathway
in
the
dynamic
EMT
and
MET
associated
with
colorectal
cancer
progression,
Clin.
Exp.
Metastasis
25
(2008)
657–663.
[76]
H.
Clevers,
Wnt/beta-catenin
signaling
in
development
and
disease,
Cell
127
(2006)
469–480.
[77]
T.
Brabletz,
A.
Jung,
S.
Reu,
M.
Porzner,
F.
Hlubek,
L.A.
Kunz-Schughart,
et
al.,
Variable
beta-catenin
expression
in
colorectal
cancers
indicates
tumor
progression
driven
by
the
tumor
environment,
Proc.
Natl.
Acad.
Sci.
U.
S.
A.
98
(2001)
10356–10361.
[78]
T.
Brabletz,
F.
Hlubek,
S.
Spaderna,
O.
Schmalhofer,
E.
Hiendlmeyer,
A.
Jung,
et
al.,
Invasion
and
metastasis
in
colorectal
cancer:
epithelial–mesenchymal
transition,
mesenchymal–epithelial
transition,
stem
cells
and
beta-catenin,
Cells
Tissues
Organs
179
(2005)
56–65.
[79]
L.
Qi,
B.
Sun,
Z.
Liu,
H.
Li,
J.
Gao,
X.
Leng,
Dickkopf-1
inhibits
epithelial–mesenchymal
transition
of
colon
cancer
cells
and
contributes
to
colon
cancer
suppression,
Cancer
Sci.
103
(2012)
828–835.
[80]
Z.Q.
Wu,
T.
Brabletz,
E.
Fearon,
A.L.
Willis,
C.Y.
Hu,
X.Y.
Li,
et
al.,
Canonical
Wnt
suppressor,
Axin2,
promotes
colon
carcinoma
oncogenic
activity,
Proc.
Natl.
Acad.
Sci.
U.
S.
A.
109
(2012)
11312–11317.
[81]
V.
Stemmer,
B.
De
Craene,
G.
Berx,
J.
Behrens,
Snail
promotes
Wnt
target
gene
expression
and
interacts
with
beta-catenin,
Oncogene
27
(2008)
5075–5080.
[82]
I.M.
Pelaez,
M.
Kalogeropoulou,
A.
Ferraro,
A.
Voulgari,
T.
Pankotai,
I.
Boros,
et
al.,
Oncogenic
RAS
alters
the
global
and
gene-specific
histone
modification
pattern
during
epithelial–mesenchymal
transition
in
colorectal
carcinoma
cells,
Int.
J.
Biochem.
Cell
Biol.
42
(2010)
911–920.
[83]
Y.
Wang,
V.N.
Ngo,
M.
Marani,
Y.
Yang,
G.
Wright,
L.M.
Staudt,
et
al.,
Critical
role
for
transcriptional
repressor
Snail2
in
transformation
by
oncogenic
RAS
in
colorectal
carcinoma
cells,
Oncogene
29
(2010)
4658–4670.
[84]
E.
Makrodouli,
E.
Oikonomou,
M.
Koc,
L.
Andera,
T.
Sasazuki,
S.
Shirasawa,
et
al.,
BRAF
and
RAS
oncogenes
regulate
Rho
GTPase
pathways
to
mediate
migration
and
invasion
properties
in
human
colon
cancer
cells:
a
comparative
study,
Mol.
Cancer
10
(2011)
118.
[85]
S.
Lemieux,
V.
Bergeron,
C.
Durand,
C.
Asselin,
N.
Saucier,
Rivard,
Constitutively
active
MEK1
is
sufficient
to
induce
epithelial-to-mesenchymal
transition
in
intestinal
epithelial
cells
and
to
promote
tumor
invasion
and
metastasis,
Int.
J.
Cancer
125
(2009)
1575–1586.
[86]
A.
Ferraro,
D.
Mourtzoukou,
V.
Kosmidou,
S.
Avlonitis,
G.
Kontogeorgos,
G.
Zografos,
et
al.,
EZH2
is
regulated
by
ERK/AKT
and
targets
integrin
alpha2
gene
to
control
epithelial–mesenchymal
transition
and
anoikis
in
colon
cancer
cells,
Int.
J.
Biochem.
Cell
Biol.
45
(2013)
243–254.
[87]
V.H.
Koelzer,
E.
Karamitopoulou,
H.
Dawson,
A.
Kondi-Pafiti,
I.
Zlobec,
A.
Lugli,
Geographic
analysis
of
RKIP
expression
and
its
clinical
relevance
in
colorectal
cancer,
Br.
J.
Cancer
108
(2013)
2088–2096.
[88]
X.
Li,
P.D.
Stevens,
J.
Liu,
H.
Yang,
W.
Wang,
C.
Wang,
et
al.,
PHLPP
is
a
negative
regulator
of
RAF1,
which
reduces
colorectal
cancer
cell
motility
and
prevents
tumor
progression
in
mice,
Gastroenterology
146
(2014)
1301–1312,
e1301–e1310.
[89]
H.K.
Roy,
B.F.
Olusola,
D.L.
Clemens,
W.J.
Karolski,
A.
Ratashak,
H.T.
Lynch,
et
al.,
AKT
proto-oncogene
overexpression
is
an
early
event
during
sporadic
colon
carcinogenesis,
Carcinogenesis
23
(2002)
201–205.
[90]
S.
Suman,
V.
Kurisetty,
T.P.
Das,
A.
Vadodkar,
G.
Ramos,
R.
Lakshmanaswamy,
et
al.,
Activation
of
AKT
signaling
promotes
epithelial–mesenchymal
transition
and
tumor
growth
in
colorectal
cancer
cells,
Mol.
Carcinog.
53
(Suppl.
1)
(2014)
E151–E160.
[91]
H.
Wang,
S.Y.
Quah,
J.M.
Dong,
E.
Manser,
J.P.
Tang,
Q.
Zeng,
PRL-3
down-regulates
PTEN
expression
and
signals
through
PI3K
to
promote
epithelial–mesenchymal
transition,
Cancer
Res.
67
(2007)
2922–2926.
[92]
P.
Gulhati,
K.A.
Bowen,
J.
Liu,
P.D.
Stevens,
P.G.
Rychahou,
M.
Chen,
et
al.,
mTORC1
and
mTORC2
regulate
EMT,
motility,
and
metastasis
of
colorectal
cancer
via
RhoA
and
Rac1
signaling
pathways,
Cancer
Res.
71
(2011)
3246–3256.
[93]
M.
Katoh,
M.
Katoh,
Cross-talk
of
WNT
and
FGF
signaling
pathways
at
GSK3beta
to
regulate
beta-catenin
and
SNAIL
signaling
cascades,
Cancer
Biol.
Ther.
5
(2006)
1059–1064.
[94]
M.L.
Slattery,
J.D.
Potter,
D.M.
Duncan,
T.D.
Berry,
Dietary
fats
and
colon
cancer:
assessment
of
risk
associated
with
specific
fatty
acids,
Int.
J.
Cancer
73
(1997)
670–677.
[95]
F.Y.
Tang,
M.H.
Pai,
E.P.
Chiang,
Consumption
of
high-fat
diet
induces
tumor
progression
and
epithelial–mesenchymal
transition
of
colorectal
cancer
in
a
mouse
xenograft
model,
J.
Nutr.
Biochem.
23
(2012)
1302–1313.
[96]
G.A.
Calin,
C.
Sevignani,
C.D.
Dumitru,
T.
Hyslop,
E.
Noch,
S.
Yendamuri,
et
al.,
Human
microRNA
genes
are
frequently
located
at
fragile
sites
and
genomic
regions
involved
in
cancers,
Proc.
Natl.
Acad.
Sci.
U.
S.
A.
101
(2004)
2999–3004.
[97]
G.J.
Hurteau,
J.A.
Carlson,
S.D.
Spivack,
G.J.
Brock,
Overexpression
of
the
microRNA
hsa-miR-200c
leads
to
reduced
expression
of
transcription
factor
8
and
increased
expression
of
E-cadherin,
Cancer
Res.
67
(2007)
7972–7976.

H.
Cao
et
al.
/
Pathology
–
Research
and
Practice
211
(2015)
557–569
567
[98]
N.R.
Christoffersen,
A.
Silahtaroglu,
U.A.
Orom,
S.
Kauppinen,
A.H.
Lund,
miR-200b
mediates
post-transcriptional
repression
of
ZFHX1B,
RNA
13
(2007)
1172–1178.
[99]
M.L.
Chen,
L.S.
Liang,
X.K.
Wang,
miR-200c
inhibits
invasion
and
migration
in
human
colon
cancer
cells
SW480/620
by
targeting
ZEB1,
Clin.
Exp.
Metastasis
29
(2012)
457–469.
[100]
C.J.
Chang,
C.H.
Chao,
W.
Xia,
J.Y.
Yang,
Y.
Xiong,
C.W.
Li,
et
al.,
p53
regulates
epithelial–mesenchymal
transition
and
stem
cell
properties
through
modulating
miRNAs,
Nat.
Cell
Biol.
13
(2011)
317–323.
[101]
P.A.
Gregory,
A.G.
Bert,
E.L.
Paterson,
S.C.
Barry,
A.
Tsykin,
G.
Farshid,
et
al.,
The
miR-200
family
and
miR-205
regulate
epithelial
to
mesenchymal
transition
by
targeting
ZEB1
and
SIP1,
Nat.
Cell
Biol.
10
(2008)
593–601.
[102]
K.
Hur,
Y.
Toiyama,
M.
Takahashi,
F.
Balaguer,
T.
Nagasaka,
J.
Koike,
et
al.,
MicroRNA-200c
modulates
epithelial-to-mesenchymal
transition
(EMT)
in
human
colorectal
cancer
metastasis,
Gut
62
(2013)
1315–1326.
[103]
U.
Burk,
J.
Schubert,
U.
Wellner,
O.
Schmalhofer,
E.
Vincan,
S.
Spaderna,
et
al.,
A
reciprocal
repression
between
ZEB1
and
members
of
the
miR-200
family
promotes
EMT
and
invasion
in
cancer
cells,
EMBO
Rep.
9
(2008)
582–589.
[104]
V.
Davalos,
C.
Moutinho,
A.
Villanueva,
R.
Boque,
P.
Silva,
F.
Carneiro,
et
al.,
Dynamic
epigenetic
regulation
of
the
microRNA-200
family
mediates
epithelial
and
mesenchymal
transitions
in
human
tumorigenesis,
Oncogene
31
(2012)
2062–2074.
[105]
E.L.
Paterson,
J.
Kazenwadel,
A.G.
Bert,
Y.
Khew-Goodall,
A.
Ruszkiewicz,
G.J.
Goodall,
Down-regulation
of
the
miRNA-200
family
at
the
invasive
front
of
colorectal
cancers
with
degraded
basement
membrane
indicates
EMT
is
involved
in
cancer
progression,
Neoplasia
15
(2013)
180–191.
[106]
L.
Bojmar,
E.
Karlsson,
S.
Ellegard,
H.
Olsson,
B.
Bjornsson,
O.
Hallbook,
et
al.,
The
role
of
microRNA-200
in
progression
of
human
colorectal
and
breast
cancer,
PLOS
ONE
8
(2013)
e84815.
[107]
M.
Pichler,
A.L.
Ress,
E.
Winter,
V.
Stiegelbauer,
M.
Karbiener,
D.
Schwarzenbacher,
et
al.,
MiR-200a
regulates
epithelial
to
mesenchymal
transition-related
gene
expression
and
determines
prognosis
in
colorectal
cancer
patients,
Br.
J.
Cancer
110
(2014)
1614–1621.
[108]
J.
Wu,
G.
Wu,
L.
Lv,
Y.F.
Ren,
X.J.
Zhang,
Y.F.
Xue,
et
al.,
MicroRNA-34a
inhibits
migration
and
invasion
of
colon
cancer
cells
via
targeting
to
Fra-1,
Carcinogenesis
33
(2012)
519–528.
[109]
H.
Siemens,
J.
Neumann,
R.
Jackstadt,
U.
Mansmann,
D.
Horst,
T.
Kirchner,
et
al.,
Detection
of
miR-34a
promoter
methylation
in
combination
with
elevated
expression
of
c-Met
and
beta-catenin
predicts
distant
metastasis
of
colon
cancer,
Clin.
Cancer
Res.
19
(2013)
710–720.
[110]
H.
Siemens,
R.
Jackstadt,
S.
Hunten,
M.
Kaller,
A.
Menssen,
U.
Gotz,
et
al.,
miR-34
and
SNAIL
form
a
double-negative
feedback
loop
to
regulate
epithelial–mesenchymal
transitions,
Cell
Cycle
10
(2011)
4256–4271.
[111]
M.
Rokavec,
M.G.
Oner,
H.
Li,
R.
Jackstadt,
L.
Jiang,
D.
Lodygin,
et
al.,
IL-6R/STAT3/miR-34a
feedback
loop
promotes
EMT-mediated
colorectal
cancer
invasion
and
metastasis,
J.
Clin.
Invest.
124
(2014)
1853–1867.
[112]
L.
Shi,
R.
Jackstadt,
H.
Siemens,
H.
Li,
T.
Kirchner,
H.
Hermeking,
p53-induced
miR-15a/16-1
and
AP4
form
a
double-negative
feedback
loop
to
regulate
epithelial–mesenchymal
transition
and
metastasis
in
colorectal
cancer,
Cancer
Res.
74
(2014)
532–542.
[113]
H.B.
Han,
J.
Gu,
H.J.
Zuo,
Z.G.
Chen,
W.
Zhao,
M.
Li,
et
al.,
Let-7c
functions
as
a
metastasis
suppressor
by
targeting
MMP11
and
PBX3
in
colorectal
cancer,
J.
Pathol.
226
(2012)
544–555.
[114]
L.
Liang,
X.
Li,
X.
Zhang,
Z.
Lv,
G.
He,
W.
Zhao,
et
al.,
MicroRNA-137,
an
HMGA1
target,
suppresses
colorectal
cancer
cell
invasion
and
metastasis
in
mice
by
directly
targeting
FMNL2,
Gastroenterology
144
(2013)
624–635,
e624.
[115]
Y.
Li,
X.
Zhu,
Y.
Zeng,
J.
Wang,
X.
Zhang,
Y.Q.
Ding,
et
al.,
FMNL2
enhances
invasion
of
colorectal
carcinoma
by
inducing
epithelial–mesenchymal
transition,
Mol.
Cancer
Res.
8
(2010)
1579–1590.
[116]
L.
Long,
G.
Huang,
H.
Zhu,
Y.
Guo,
Y.
Liu,
J.
Huo,
Down-regulation
of
miR-138
promotes
colorectal
cancer
metastasis
via
directly
targeting
TWIST2,
J.
Transl.
Med.
11
(2013)
275.
[117]
X.
Meng,
J.
Wu,
C.
Pan,
H.
Wang,
X.
Ying,
Y.
Zhou,
et
al.,
Genetic
and
epigenetic
down-regulation
of
microRNA-212
promotes
colorectal
tumor
metastasis
via
dysregulation
of
MnSOD,
Gastroenterology
145
(2013)
426–436,
e421–e426.
[118]
H.
Zhao,
Z.
Xu,
H.
Qin,
Z.
Gao,
L.
Gao,
miR-30b
regulates
migration
and
invasion
of
human
colorectal
cancer
via
SIX1,
Biochem.
J.
460
(2014)
117–125.
[119]
H.
Zhao,
T.
Dong,
H.
Zhou,
L.
Wang,
A.
Huang,
B.
Feng,
et
al.,
miR-320a
suppresses
colorectal
cancer
progression
by
targeting
Rac1,
Carcinogenesis
35
(2014)
886–895.
[120]
A.
Strillacci,
M.C.
Valerii,
P.
Sansone,
C.
Caggiano,
A.
Sgromo,
L.
Vittori,
et
al.,
Loss
of
miR-101
expression
promotes
Wnt/beta-catenin
signalling
pathway
activation
and
malignancy
in
colon
cancer
cells,
J.
Pathol.
229
(2013)
379–389.
[121]
S.
Pizzini,
A.
Bisognin,
S.
Mandruzzato,
M.
Biasiolo,
A.
Facciolli,
L.
Perilli,
et
al.,
Impact
of
microRNAs
on
regulatory
networks
and
pathways
in
human
colorectal
carcinogenesis
and
development
of
metastasis,
BMC
Genomics
14
(2013)
589.
[122]
C.L.
Cottonham,
S.
Kaneko,
L.
Xu,
miR-21
and
miR-31
converge
on
TIAM1
to
regulate
migration
and
invasion
of
colon
carcinoma
cells,
J.
Biol.
Chem.
285
(2010)
35293–35302.
[123]
A.
Ferraro,
C.K.
Kontos,
T.
Boni,
I.
Bantounas,
D.
Siakouli,
V.
Kosmidou,
et
al.,
Epigenetic
regulation
of
miR-21
in
colorectal
cancer:
ITGB4
as
a
novel
miR-21
target
and
a
three-gene
network
(miR-21-ITGBeta4-PDCD4)
as
predictor
of
metastatic
tumor
potential,
Epigenetics
9
(2014)
129–141.
[124]
G.H.
Liu,
Z.G.
Zhou,
R.
Chen,
M.J.
Wang,
B.
Zhou,
Y.
Li,
et
al.,
Serum
miR-21
and
miR-92a
as
biomarkers
in
the
diagnosis
and
prognosis
of
colorectal
cancer,
Tumour
Biol.
34
(2013)
2175–2181.
[125]
X.
Xia,
B.
Yang,
X.
Zhai,
X.
Liu,
K.
Shen,
Z.
Wu,
et
al.,
Prognostic
role
of
microRNA-21
in
colorectal
cancer:
a
meta-analysis,
PLOS
ONE
8
(2013)
e80426.
[126]
M.H.
Yang,
J.
Yu,
N.
Chen,
X.Y.
Wang,
X.Y.
Liu,
S.
Wang,
et
al.,
Elevated
microRNA-31
expression
regulates
colorectal
cancer
progression
by
repressing
its
target
gene
SATB2,
PLOS
ONE
8
(2013)
e85353.
[127]
T.
Colangelo,
A.
Fucci,
C.
Votino,
L.
Sabatino,
M.
Pancione,
C.
Laudanna,
et
al.,
MicroRNA-130b
promotes
tumor
development
and
is
associated
with
poor
prognosis
in
colorectal
cancer,
Neoplasia
15
(2013)
1218–1231.
[128]
W.
Tang,
Y.
Zhu,
J.
Gao,
J.
Fu,
C.
Liu,
Y.
Liu,
et
al.,
MicroRNA-29a
promotes
colorectal
cancer
metastasis
by
regulating
matrix
metalloproteinase
2
and
E-cadherin
via
KLF4,
Br.
J.
Cancer
110
(2014)
450–458.
[129]
H.Y.
Chen,
Y.M.
Lin,
H.C.
Chung,
Y.D.
Lang,
C.J.
Lin,
J.
Huang,
et
al.,
miR-103/107
promote
metastasis
of
colorectal
cancer
by
targeting
the
metastasis
suppressors
DAPK
and
KLF4,
Cancer
Res.
72
(2012)
3631–3641.
[130]
U.A.
Orom,
T.
Derrien,
M.
Beringer,
K.
Gumireddy,
A.
Gardini,
G.
Bussotti,
et
al.,
Long
noncoding
RNAs
with
enhancer-like
function
in
human
cells,
Cell
143
(2010)
46–58.
[131]
C.
Padua
Alves,
A.S.
Fonseca,
B.R.
Muys,
E.L.B.R.
De
Barros,
M.C.
Burger,
J.E.
De
Souza,
et
al.,
Brief
report:
the
lincRNA
hotair
is
required
for
epithelial-to-mesenchymal
transition
and
stemness
maintenance
of
cancer
cell
lines,
Stem
Cells
31
(2013)
2827–2832.
[132]
Y.
Fan,
B.
Shen,
M.
Tan,
X.
Mu,
Y.
Qin,
F.
Zhang,
et
al.,
TGF-beta-induced
upregulation
of
malat1
promotes
bladder
cancer
metastasis
by
associating
with
suz12,
Clin.
Cancer
Res.
20
(2014)
1531–1541.
[133]
J.H.
Yuan,
F.
Yang,
F.
Wang,
J.Z.
Ma,
Y.J.
Guo,
Q.F.
Tao,
et
al.,
A
long
noncoding
RNA
activated
by
TGF-beta
promotes
the
invasion-metastasis
cascade
in
hepatocellular
carcinoma,
Cancer
Cell
25
(2014)
666–681.
[134]
G.S.
Karagiannis,
T.
Poutahidis,
S.E.
Erdman,
R.
Kirsch,
R.H.
Riddell,
E.P.
Diamandis,
Cancer-associated
fibroblasts
drive
the
progression
of
metastasis
through
both
paracrine
and
mechanical
pressure
on
cancer
tissue,
Mol.
Cancer
Res.
10
(2012)
1403–1418.
[135]
M.
Herrera,
A.B.
Islam,
A.
Herrera,
P.
Martin,
V.
Garcia,
J.
Silva,
et
al.,
Functional
heterogeneity
of
cancer-associated
fibroblasts
from
human
colon
tumors
shows
specific
prognostic
gene
expression
signature,
Clin.
Cancer
Res.
19
(2013)
5914–5926.
[136]
G.S.
Karagiannis,
C.
Petraki,
I.
Prassas,
P.
Saraon,
N.
Musrap,
A.
Dimitromanolakis,
et
al.,
Proteomic
signatures
of
the
desmoplastic
invasion
front
reveal
collagen
type
XII
as
a
marker
of
myofibroblastic
differentiation
during
colorectal
cancer
metastasis,
Oncotarget
3
(2012)
267–285.
[137]
C.
Rupp,
M.
Scherzer,
A.
Rudisch,
C.
Unger,
C.
Haslinger,
N.
Schweifer,
et
al.,
IGFBP7,
a
novel
tumor
stroma
marker,
with
growth-promoting
effects
in
colon
cancer
through
a
paracrine
tumor–stroma
interaction,
Oncogene
34
(2015)
815–825.
[138]
S.
Zhu,
J.
Zhang,
F.
Xu,
E.
Xu,
W.
Ruan,
Y.
Ma,
et
al.,
IGFBP-rP1
suppresses
epithelial–mesenchymal
transition
and
metastasis
in
colorectal
cancer,
Cell
Death
Dis.
6
(2015)
e1695.
[139]
S.A.
Jensen,
B.
Vainer,
A.
Bartels,
N.
Brunner,
J.B.
Sorensen,
Expression
of
matrix
metalloproteinase
9
(MMP-9)
and
tissue
inhibitor
of
metalloproteinases
1
(TIMP-1)
by
colorectal
cancer
cells
and
adjacent
stroma
cells
–
associations
with
histopathology
and
patients
outcome,
Eur.
J.
Cancer
46
(2010)
3233–3242.
[140]
Y.
Li,
L.
Wang,
L.
Pappan,
A.
Galliher-Beckley,
J.
Shi,
IL-1beta
promotes
stemness
and
invasiveness
of
colon
cancer
cells
through
Zeb1
activation,
Mol.
Cancer
11
(2012)
87.
[141]
X.S.
Cheng,
Y.F.
Li,
J.
Tan,
B.
Sun,
Y.C.
Xiao,
X.B.
Fang,
et
al.,
CCL20
and
CXCL8
synergize
to
promote
progression
and
poor
survival
outcome
in
patients
with
colorectal
cancer
by
collaborative
induction
of
the
epithelial–mesenchymal
transition,
Cancer
Lett.
348
(2014)
77–87.
[142]
S.
Matsumoto,
T.
Hara,
K.
Mitsuyama,
M.
Yamamoto,
O.
Tsuruta,
M.
Sata,
et
al.,
Essential
roles
of
IL-6
trans-signaling
in
colonic
epithelial
cells,
induced
by
the
IL-6/soluble-IL-6
receptor
derived
from
lamina
propria
macrophages,
on
the
development
of
colitis-associated
premalignant
cancer
in
a
murine
model,
J.
Immunol.
184
(2010)
1543–1551.
[143]
E.
Brighenti,
C.
Calabrese,
G.
Liguori,
F.A.
Giannone,
D.
Trere,
L.
Montanaro,
et
al.,
Interleukin
6
downregulates
p53
expression
and
activity
by
stimulating
ribosome
biogenesis:
a
new
pathway
connecting
inflammation
to
cancer,
Oncogene
33
(2014)
4396–4406.
[144]
H.
Liu,
G.
Ren,
T.
Wang,
Y.
Chen,
C.
Gong,
Y.
Bai,
et
al.,
Aberrantly
expressed
Fra-1
by
IL-6/STAT3
transactivation
promotes
colorectal
cancer
aggressiveness
through
epithelial–mesenchymal
transition,
Carcinogenesis
36
(2015)
459–468.
[145]
S.P.
Chellappan,
H.
Wang,
H.-S.
Wang,
B.-H.
Zhou,
C.-L.
Li,
F.
Zhang,
et
al.,
Epithelial–mesenchymal
transition
(EMT)
induced
by
TNF-␣
requires
AKT/GSK-3␤-mediated
stabilization
of
snail
in
colorectal
cancer,
PLOS
ONE
8
(2013)
e56664.
[146]
R.C.
Bates,
M.J.
Deleo,
A.M.
Mercurio
3rd,
The
epithelial–mesenchymal
transition
of
colon
carcinoma
involves
expression
of
IL-8
and

568
H.
Cao
et
al.
/
Pathology
–
Research
and
Practice
211
(2015)
557–569
CXCR-1-mediated
chemotaxis,
Exp.
Cell
Res.
299
(2004)
315–324.
[147]
S.
Torres,
R.A.
Bartolome,
M.
Mendes,
R.
Barderas,
M.J.
Fernandez-Acenero,
A.
Pelaez-Garcia,
et
al.,
Proteome
profiling
of
cancer-associated
fibroblasts
identifies
novel
proinflammatory
signatures
and
prognostic
markers
for
colorectal
cancer,
Clin.
Cancer
Res.
19
(2013)
6006–6019.
[148]
A.
Herrera,
M.
Herrera,
L.
Alba-Castellon,
J.
Silva,
V.
Garcia,
J.
Loubat-Casanovas,
et
al.,
Protumorigenic
effects
of
Snail-expression
fibroblasts
on
colon
cancer
cells,
Int.
J.
Cancer
134
(2014)
2984–2990.
[149]
W.L.
Hwang,
M.H.
Yang,
M.L.
Tsai,
H.Y.
Lan,
S.H.
Su,
S.C.
Chang,
et
al.,
SNAIL
regulates
interleukin-8
expression,
stem
cell-like
activity,
and
tumorigenicity
of
human
colorectal
carcinoma
cells,
Gastroenterology
141
(2011)
279–291,
291
e271–e275.
[150]
X.
Zeng,
P.
Yang,
B.
Chen,
X.
Jin,
Y.
Liu,
X.
Zhao,
et
al.,
Quantitative
secretome
analysis
reveals
the
interactions
between
epithelia
and
tumor
cells
by
in
vitro
modulating
colon
cancer
microenvironment,
J.
Proteom.
89
(2013)
51–70.
[151]
Y.
Itatani,
K.
Kawada,
T.
Fujishita,
F.
Kakizaki,
H.
Hirai,
T.
Matsumoto,
et
al.,
Loss
of
SMAD4
from
colorectal
cancer
cells
promotes
CCL15
expression
to
recruit
CCR1+
myeloid
cells
and
facilitate
liver
metastasis,
Gastroenterology
145
(2013)
1064–1075,
e1011.
[152]
T.
Kitamura,
T.
Fujishita,
P.
Loetscher,
L.
Revesz,
H.
Hashida,
S.
Kizaka-Kondoh,
et
al.,
Inactivation
of
chemokine
(C–C
motif)
receptor
1
(CCR1)
suppresses
colon
cancer
liver
metastasis
by
blocking
accumulation
of
immature
myeloid
cells
in
a
mouse
model,
Proc.
Natl.
Acad.
Sci.
U.
S.
A.
107
(2010)
13063–13068.
[153]
V.
Mele,
M.G.
Muraro,
D.
Calabrese,
D.
Pfaff,
N.
Amatruda,
F.
Amicarella,
et
al.,
Mesenchymal
stromal
cells
induce
epithelial-to-mesenchymal
transition
in
human
colorectal
cancer
cells
through
the
expression
of
surface-bound
TGF-beta,
Int.
J.
Cancer
134
(2014)
2583–2594.
[154]
L.J.
Gay,
B.
Felding-Habermann,
Contribution
of
platelets
to
tumour
metastasis,
Nat.
Rev.
Cancer
11
(2011)
123–134.
[155]
M.
Labelle,
S.
Begum,
R.O.
Hynes,
Direct
signaling
between
platelets
and
cancer
cells
induces
an
epithelial–mesenchymal-like
transition
and
promotes
metastasis,
Cancer
Cell
20
(2011)
576–590.
[156]
E.J.
Steller,
D.A.
Raats,
J.
Koster,
B.
Rutten,
K.M.
Govaert,
B.L.
Emmink,
et
al.,
PDGFRB
promotes
liver
metastasis
formation
of
mesenchymal-like
colorectal
tumor
cells,
Neoplasia
15
(2013)
204–217.
[157]
J.
Ou,
Y.
Peng,
J.
Deng,
H.
Miao,
J.
Zhou,
L.
Zha,
et
al.,
Endothelial
cell-derived-fibronectin
extra
domain
A
promotes
colorectal
cancer
metastasis
via
inducing
epithelial–mesenchymal
transition,
Carcinogenesis
35
(2014)
1661–1670.
[158]
F.
Prall,
Tumour
budding
in
colorectal
carcinoma,
Histopathology
50
(2007)
151–162.
[159]
H.
Ueno,
J.
Murphy,
J.R.
Jass,
H.
Mochizuki,
I.C.
Talbot,
Tumour
‘budding’
as
an
index
to
estimate
the
potential
of
aggressiveness
in
rectal
cancer,
Histopathology
40
(2002)
127–132.
[160]
K.
Hase,
C.
Shatney,
D.
Johnson,
M.
Trollope,
M.
Vierra,
Prognostic
value
of
tumor
“budding”
in
patients
with
colorectal
cancer,
Dis.
Colon
Rectum
36
(1993)
627–635.
[161]
T.
Nakamura,
H.
Mitomi,
S.
Kikuchi,
Y.
Ohtani,
K.
Sato,
Evaluation
of
the
usefulness
of
tumor
budding
on
the
prediction
of
metastasis
to
the
lung
and
liver
after
curative
excision
of
colorectal
cancer,
Hepatogastroenterology
52
(2005)
1432–1435.
[162]
L.M.
Wang,
D.
Kevans,
H.
Mulcahy,
J.
O’sullivan,
D.
Fennelly,
J.
Hyland,
et
al.,
Tumor
budding
is
a
strong
and
reproducible
prognostic
marker
in
T3N0
colorectal
cancer,
Am.
J.
Surg.
Pathol.
33
(2009)
134–141.
[163]
M.
Tanaka,
Y.
Hashiguchi,
H.
Ueno,
K.
Hase,
H.
Mochizuki,
Tumor
budding
at
the
invasive
margin
can
predict
patients
at
high
risk
of
recurrence
after
curative
surgery
for
stage
II,
T3
colon
cancer,
Dis.
Colon
Rectum
46
(2003)
1054–1059.
[164]
F.
Prall,
H.
Nizze,
M.
Barten,
Tumour
budding
as
prognostic
factor
in
stage
I/II
colorectal
carcinoma,
Histopathology
47
(2005)
17–24.
[165]
C.
Compton,
Prognostic
Factors
in
Cancer.
3rd
edn.
Wiley-Liss,
New
York.
2006.
[166]
T.
Kirchner,
T.
Brabletz,
Patterning
and
nuclear
beta-catenin
expression
in
the
colonic
adenoma–carcinoma
sequence.
Analogies
with
embryonic
gastrulation,
Am.
J.
Pathol.
157
(2000)
1113–1121.
[167]
D.T.
Khanh,
E.
Mekata,
K.-I.
Mukaisho,
H.
Sugihara,
T.
Shimizu,
H.
Shiomi,
et
al.,
Prognostic
role
of
CD10+
myeloid
cells
in
association
with
tumor
budding
at
the
invasion
front
of
colorectal
cancer,
Cancer
Sci.
102
(2011)
1724–1733.
[168]
R.
Oshiro,
H.
Yamamoto,
H.
Takahashi,
M.
Ohtsuka,
X.
Wu,
J.
Nishimura,
et
al.,
C4.4A
is
associated
with
tumor
budding
and
epithelial–mesenchymal
transition
of
colorectal
cancer,
Cancer
Sci.
103
(2012)
1155–1164.
[169]
K.
Guzinska-Ustymowicz,
MMP-9
and
cathepsin
B
expression
in
tumor
budding
as
an
indicator
of
a
more
aggressive
phenotype
of
colorectal
cancer
(CRC),
Anticancer
Res.
26
(2006)
1589–1594.
[170]
B.
Markl,
I.
Renk,
D.V.
Oruzio,
H.
Jahnig,
G.
Schenkirsch,
C.
Scholer,
et
al.,
Tumour
budding,
uPA
and
PAI-1
are
associated
with
aggressive
behaviour
in
colon
cancer,
J.
Surg.
Oncol.
102
(2010)
235–241.
[171]
T.
Masaki,
M.
Sugiyama,
H.
Matsuoka,
N.
Abe,
Y.
Izumisato,
A.
Sakamoto,
et
al.,
Coexpression
of
matrilysin
and
laminin-5
gamma2
chain
may
contribute
to
tumor
cell
migration
in
colorectal
carcinomas,
Dig.
Dis.
Sci.
48
(2003)
1262–1267.
[172]
I.
Zlobec,
A.
Lugli,
Epithelial–mesenchymal
transition
and
tumor
budding
in
aggressive
colorectal
cancer:
tumor
budding
as
oncotarget,
Oncotarget
1
(2010)
651–661.
[173]
A.D.
Yang,
Chronic
oxaliplatin
resistance
induces
epithelial-to-mesenchymal
transition
in
colorectal
cancer
cell
lines,
Clin.
Cancer
Res.
12
(2006)
4147–4153.
[174]
H.X.
Zheng,
Y.D.
Cai,
Y.D.
Wang,
X.B.
Cui,
T.T.
Xie,
W.J.
Li,
et
al.,
Fas
signaling
promotes
motility
and
metastasis
through
epithelial–mesenchymal
transition
in
gastrointestinal
cancer,
Oncogene
32
(2013)
1183–1192.
[175]
H.
Hoshino,
N.
Miyoshi,
K.
Nagai,
Y.
Tomimaru,
H.
Nagano,
M.
Sekimoto,
et
al.,
Epithelial–mesenchymal
transition
with
expression
of
SNAI1-induced
chemoresistance
in
colorectal
cancer,
Biochem.
Biophys.
Res.
Commun.
390
(2009)
1061–1065.
[176]
V.J.
Findlay,
R.E.
Moretz,
C.
Wang,
S.G.
Vaena,
S.G.
Bandurraga,
M.
Ashenafi,
et
al.,
Slug
expression
inhibits
calcitriol-mediated
sensitivity
to
radiation
in
colorectal
cancer,
Mol.
Carcinog.
53
(Suppl.
1)
(2014)
E130–E139.
[177]
A.
Albasri,
R.
Seth,
D.
Jackson,
A.
Benhasouna,
S.
Crook,
A.S.
Nateri,
et
al.,
C-terminal
Tensin-like
(CTEN)
is
an
oncogene
which
alters
cell
motility
possibly
through
repression
of
E-cadherin
in
colorectal
cancer,
J.
Pathol.
218
(2009)
57–65.
[178]
D.R.
Cochrane,
N.S.
Spoelstra,
E.N.
Howe,
S.K.
Nordeen,
J.K.
Richer,
MicroRNA-200c
mitigates
invasiveness
and
restores
sensitivity
to
microtubule-targeting
chemotherapeutic
agents,
Mol.
Cancer
Ther.
8
(2009)
1055–1066.
[179]
X.
Chen,
Y.
Wang,
H.
Xia,
Q.
Wang,
X.
Jiang,
Z.
Lin,
et
al.,
Loss
of
E-cadherin
promotes
the
growth,
invasion
and
drug
resistance
of
colorectal
cancer
cells
and
is
associated
with
liver
metastasis,
Mol.
Biol.
Rep.
39
(2012)
6707–6714.
[180]
A.
Kawamoto,
T.
Yokoe,
K.
Tanaka,
S.
Saigusa,
Y.
Toiyama,
H.
Yasuda,
et
al.,
Radiation
induces
epithelial–mesenchymal
transition
in
colorectal
cancer
cells,
Oncol.
Rep.
27
(2012)
51–57.
[181]
C.
Kudo-Saito,
H.
Shirako,
T.
Takeuchi,
Y.
Kawakami,
Cancer
metastasis
is
accelerated
through
immunosuppression
during
Snail-induced
EMT
of
cancer
cells,
Cancer
Cell
15
(2009)
195–206.
[182]
E.
Buck,
A.
Eyzaguirre,
S.
Barr,
S.
Thompson,
R.
Sennello,
D.
Young,
et
al.,
Loss
of
homotypic
cell
adhesion
by
epithelial–mesenchymal
transition
or
mutation
limits
sensitivity
to
epidermal
growth
factor
receptor
inhibition,
Mol.
Cancer
Ther.
6
(2007)
532–541.
[183]
A.
Trumpp,
O.D.
Wiestler,
Mechanisms
of
disease:
cancer
stem
cells
–
targeting
the
evil
twin,
Nat.
Clin.
Pract.
Oncol.
5
(2008)
337–347.
[184]
C.W.
Fan,
T.
Chen,
Y.N.
Shang,
Y.Z.
Gu,
S.L.
Zhang,
R.
Lu,
et
al.,
Cancer-initiating
cells
derived
from
human
rectal
adenocarcinoma
tissues
carry
mesenchymal
phenotypes
and
resist
drug
therapies,
Cell
Death
Dis.
4
(2013)
e828.
[185]
X.
Li,
M.T.
Lewis,
J.
Huang,
C.
Gutierrez,
C.K.
Osborne,
M.F.
Wu,
et
al.,
Intrinsic
resistance
of
tumorigenic
breast
cancer
cells
to
chemotherapy,
J.
Natl.
Cancer
Inst.
100
(2008)
672–679.
[186]
T.
Hirano,
R.
Satow,
A.
Kato,
M.
Tamura,
Y.
Murayama,
H.
Saya,
et
al.,
Identification
of
novel
small
compounds
that
restore
E-cadherin
expression
and
inhibit
tumor
cell
motility
and
invasiveness,
Biochem.
Pharmacol.
86
(2013)
1419–1429.
[187]
R.I.
Fernando,
M.
Litzinger,
P.
Trono,
D.H.
Hamilton,
J.
Schlom,
C.
Palena,
The
T-box
transcription
factor
Brachyury
promotes
epithelial–mesenchymal
transition
in
human
tumor
cells,
J.
Clin.
Invest.
120
(2010)
533–544.
[188]
M.
Roselli,
R.I.
Fernando,
F.
Guadagni,
A.
Spila,
J.
Alessandroni,
R.
Palmirotta,
et
al.,
Brachyury,
a
driver
of
the
epithelial–mesenchymal
transition,
is
overexpressed
in
human
lung
tumors:
an
opportunity
for
novel
interventions
against
lung
cancer,
Clin.
Cancer
Res.
18
(2012)
3868–3879.
[189]
C.
Palena,
D.E.
Polev,
K.Y.
Tsang,
R.I.
Fernando,
M.
Litzinger,
L.L.
Krukovskaya,
et
al.,
The
human
T-box
mesodermal
transcription
factor
Brachyury
is
a
candidate
target
for
T-cell-mediated
cancer
immunotherapy,
Clin.
Cancer
Res.
13
(2007)
2471–2478.
[190]
D.H.
Hamilton,
M.T.
Litzinger,
A.
Jales,
B.
Huang,
R.I.
Fernando,
J.W.
Hodge,
et
al.,
Immunological
targeting
of
tumor
cells
undergoing
an
epithelial–mesenchymal
transition
via
a
recombinant
brachyury–yeast
vaccine,
Oncotarget
4
(2013)
1777–1790.
[191]
A.
Ardiani,
S.R.
Gameiro,
C.
Palena,
D.H.
Hamilton,
A.
Kwilas,
T.H.
King,
et
al.,
Vaccine-mediated
immunotherapy
directed
against
a
transcription
factor
driving
the
metastatic
process,
Cancer
Res.
74
(2014)
1945–1957.
[192]
S.
Vidic,
B.
Markelc,
G.
Sersa,
A.
Coer,
U.
Kamensek,
G.
Tevz,
et
al.,
MicroRNAs
targeting
mutant
K-ras
by
electrotransfer
inhibit
human
colorectal
adenocarcinoma
cell
growth
in
vitro
and
in
vivo,
Cancer
Gene
Ther.
17
(2010)
409–419.
[193]
K.N.
Chua,
W.J.
Sim,
V.
Racine,
S.Y.
Lee,
B.C.
Goh,
J.P.
Thiery,
A
cell-based
small
molecule
screening
method
for
identifying
inhibitors
of
epithelial–mesenchymal
transition
in
carcinoma,
PLoS
ONE
7
(2012)
e33183.
[194]
S.A.
Flanigan,
T.M.
Pitts,
T.P.
Newton,
G.N.
Kulikowski,
A.C.
Tan,
M.C.
Mcmanus,
et
al.,
Overcoming
IGF1R/IR
resistance
through
inhibition
of
MEK
signaling
in
colorectal
cancer
models,
Clin.
Cancer
Res.
19
(2013)
6219–6229.
[195]
C.
Bocca,
F.
Bozzo,
S.
Cannito,
M.
Parola,
A.
Miglietta,
Celecoxib
inactivates
epithelial–mesenchymal
transition
stimulated
by
hypoxia
and/or
epidermal
growth
factor
in
colon
cancer
cells,
Mol.
Carcinog.
51
(2012)
783–795.

H.
Cao
et
al.
/
Pathology
–
Research
and
Practice
211
(2015)
557–569
569
[196]
A.K.
Reka,
R.
Kuick,
H.
Kurapati,
T.J.
Standiford,
G.S.
Omenn,
V.G.
Keshamouni,
Identifying
inhibitors
of
epithelial–mesenchymal
transition
by
connectivity
map-based
systems
approach,
J.
Thorac.
Oncol.
6
(2011)
1784–1792.
[197]
I.G.
Zubeldia,
A.M.
Bleau,
M.
Redrado,
D.
Serrano,
A.
Agliano,
C.
Gil-Puig,
et
al.,
Epithelial
to
mesenchymal
transition
and
cancer
stem
cell
phenotypes
leading
to
liver
metastasis
are
abrogated
by
the
novel
TGFbeta1-targeting
peptides
P17
and
P144,
Exp.
Cell
Res.
319
(2013)
12–22.
[198]
P.B.
Gupta,
T.T.
Onder,
G.
Jiang,
K.
Tao,
C.
Kuperwasser,
R.A.
Weinberg,
et
al.,
Identification
of
selective
inhibitors
of
cancer
stem
cells
by
high-throughput
screening,
Cell
138
(2009)
645–659.
[199]
John
Giannios,
et
al.,
Inhibition
of
epithelial–mesenchymal
transition
(EMT)
with
immunochemogene
treatment
in
metastatic
colorectal
cancer,
Gastrointest.
Cancer
Res.
Suppl2
(2010)
S11.
[200]
R.
Harouaka,
Z.
Kang,
S.Y.
Zheng,
L.
Cao,
Circulating
tumor
cells:
advances
in
isolation
and
analysis,
and
challenges
for
clinical
applications,
Pharmacol.
Ther.
141
(2014)
209–221.
[201]
A.D.
Rhim,
E.T.
Mirek,
N.M.
Aiello,
A.
Maitra,
J.M.
Bailey,
F.
Mcallister,
et
al.,
EMT
and
dissemination
precede
pancreatic
tumor
formation,
Cell
148
(2012)
349–361.