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Crosstalk between TGF-b signaling andthe microRNA machinery
Henriett Butz1, Karoly Racz1, LaszloHunyady2 and Attila Patocs3,412nd
Department
of
Medicine,
Faculty
of
Medicine,
Semmelweis
University,
Budapest,
Hungary2Department
of
Physiology,
Semmelweis
University,
Budapest,
Hungary3Molecular
Medicine
Research
Group,
Hungarian
Academy
of
Sciences,
Budapest,
Hungary4Department
of
Laboratory
Medicine,
Semmelweis
University,
Budapest,
Hungary
The activin/transforming growth factor-b (TGF-b) path-
way
plays
an
important
role
in
tumorigenesis
either
by
its tumor suppressor or tumor promoting effect. Loss of
members of the TGF-b signaling by somatic mutations
or
epigenetic
events,
such
as
DNA
methylation
or
regu-
lation by microRNA (miRNA), may affect the signaling
process. Most members of the TGF-b pathway are
known to be targeted by one or more miRNAs. In addi-
tion, the biogenesis of miRNAs is also regulated by TGF-
b both directly and through SMADs. Based on these
interactions, it appears that autoregulatory feedback
loops between TGF-b and miRNAs influence the fate
of tumor cells. Our aim is to review the crosstalk be-
tween TGF-b signaling and the miRNA machinery to
highlight
potential
novel
therapeutic
targets.
Members and functions of the TGF-b signaling pathway
The activin/TGF-b family
consists
of
evolutionarily
con-
served
polypeptides,
which
play
prominent
roles
in the
regulation
of
embryonic
development,
reproduction
andtumor
formation
[1]. TGF-b signaling
is
often
considered
a pathogenic factor in tumorigenesis due to its tumor
suppressor
or
tumor
promoting
effects
(see
below).
Altered
TGF-b signaling has been frequently shown in different
tumor
types,
including
breast,
prostate,
endometrial,
colo-
rectal,
thyroid,
parathyroid
and
pancreas
neoplasms
[2].
The TGF-b family comprises more than 35 members,
including
TGF-bs, bone
morphogenetic
proteins
(BMPs),
growth
differentiation
factors
(GDFs),
activins,
inhibins,
Mullerian inhibiting substance (MIS), Nodal and leftys [1].
They
are
secreted
in
an
inactive
form,
and
TGF-bs
become
active
after
cleavage
of
the
N-terminal
pro-region,
referred
to
as
the
latency-associated
peptide
(LAP).
Until
thiscleavage occurs, the LAP-associated TGF-bs (L-TGF-bs)
cannot interact
with
their
receptors,
and
hence
they
are
biologically
inactive
[1]. The
active
TGF-b
molecule
is
a
dimer composed of two TGF-b molecules linked by a disul-
fide
bridge
between
the
ninth
cysteine
of
each
monomer
[1].
TGF-b
receptors
are
serine/threonine
kinase
receptors
and are divided into three groups: type I, type II and type
III. In
mammals
there
are
seven
different
members
of
type
I (ALK1ALK7)
and
five
members
of
type
II receptors
(ACVR2, ACVR2B, AMHR2, BMPR2, TGF-bR2). Upon
ligand
binding,
an
active
ligand-type
I/type
II
receptor
complex is formed, type II receptors activate type I recep-
tors
by
phosphorylation,
and
type
I
receptors
subsequently
phosphorylate
downstream
SMAD
proteins
which
trans-
mit the signal to the nucleus (Figure 1). Type III receptors
(betaglycan
and
endoglin)
have
a
higher
molecular
weight
than
type
I
and
type
II receptors.
Betaglycan
is
a
mem-
brane-anchored proteoglycan that can bind TGF-bs and
facilitates
interaction
of
TGF-b-type
II
receptor
with
TGF-
b [1]. It can also promote binding of inhibin to type II
receptor,
thereby
antagonizing
activin
signaling.
Endoglin
is a
membrane
glycoprotein
with
a
large
extracellular
domain containing an integrin recognition motif and a
short
cytoplasmic
tail
with
serine
and
threonine
residues,
which can be phosphorylated by the TGF-b receptors.
Endoglin
phosphorylation
seems
to
play
a
regulatory
role
for
ALK1-dependent
endothelial
cell
growth
and
adhesion,
which is confirmed by the findings that endoglin was found
to be
overexpressed
in several
tumor
types
[3,4].
Tumor suppressor and tumor promoting effects of theTGF-b pathway
TGF-b
may
inhibit
cell
proliferation
at
multiple
levels.
Well-known
tumor
suppressors,
such
as
p15Ink4b and
p21Waf1 are
induced
by
TGF-b
signaling,
whereas
oncogen-
ic
factors
such
as
c-Myc,
a
transcription
factor
that
pro-
motes cell proliferation, and Id proteins, nuclear factors
that
inhibit
cell
differentiation,
are
repressed
via
TGF-b
signaling
[2].
TGF-b
can
also
activate
apoptosis
[5].
This
function is mediated by its downstream targets, such as
death-associated
protein
kinase
(DAPK),
growth
arrest
and DNA-damage
inducible
b
(GADD45b),
and
Bcl2-like
11 (BCL2L11 or BIM). TGF-b signaling also inhibits tumor
growth
by
repressing
hepatocyte
growth
factor
(HGF),macrophage-stimulating protein (MSP) and TGF-a [2].
In
addition
to
its
tumor
suppressor
effects,
TGF-b
sig-
naling has tumor promoting downstream targets. For
example,
through
induction
of
deleted
in
esophageal
can-
cer
1
(DEC1),
platelet-derived
growth
factor
beta
polypep-
tide (PDGF-B), protein snail homolog 1 (SNAIL) and high
mobility
group
AT-hook
2
(HMGA2),
TGF-b
signaling
may
mediate
antiapoptotic
effects,
growth
stimulation
and
epi-
thelialmesenchymal transition (EMT). EMT is a biologi-
cal
process
through
which
a
polarized
cell
that
normally
interacts with a basement membrane (epithelial pheno-
type) switches to a mesenchymal phenotype that is char-
acterized by invasiveness and increased cell mobility [6].
Review
Corresponding author: Patocs, A. (patatt@bel2.sote.hu).
Keywords: TGF-b signaling pathway; miRNA; endocrine neoplasm.
382 0165-6147/$ see front matter 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tips.2012.04.003 Trends in Pharmacological Sciences, July 2012, Vol. 33, No. 7
mailto:patatt@bel2.sote.huhttp://dx.doi.org/10.1016/j.tips.2012.04.003http://dx.doi.org/10.1016/j.tips.2012.04.003mailto:patatt@bel2.sote.hu7/24/2019 Crosstalk Between TGF-b Signaling
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During this process, basement membrane degradation
occurs,
cells
lose
E-cadherin
and
produce
vimentin,
a
mesenchymal
cell-specific
intermediate
filament.
EMT
has key roles in embryogenesis and wound healing, and
has
also
been
described
as
a
crucial
mechanism
for
the
acquisition
of
malignant
phenotypes
of
epithelial
cells
[7].
Various factors are reported to mediate TGF-b-induced
EMT,
including
SNAIL1,
SNAIL2
(Slug),
ZEB1,
ZEB2
(SIP1).
These
are
transcriptional
repressors
that
bind
to
E-box motifs of the DNA and repress transcription of
various
genes,
including
E-cadherin
[2].
It
is
generally accepted that TGF-b signaling
exerts
tumor suppressor
effects
during
the early
phase
of
tumori-
genesis and, in certain situations, it switches from a
tumor suppressor
to
a
tumor promoter
[2].
For the tumor
promoting effect, additional secondary molecular mecha-
nisms,
includingmutation
of
theTP53
gene
encodingtumor
suppressor
p53
[8] or
Ras
activation [9],
are also involved.
Ras enhances induction of SNAIL by TGF-b; however, this
process
is
also regulated by
glycogen
synthase kinase-3b
(GSK-3b).
GSK-3b is
apparently
one of
themajor
mediators
of environment-dependent TGF-b-induced responses, as a
downstream
collector
of
multiple signaling
pathways,
in-
cluding mitogen-activated
protein
kinase
(MAPK),
PI3K/
Akt and Wnt [2].
In
addition
to
the effects of genes encoding members
of the TGF-b signaling, regulation
of
TGF-b expression
by epigenetic mechanisms
via DNA
methylation or
miR-
NAs has also been demonstrated in different tumors
[10,11].
TGF-/activin
TGFBR II TGFBR I
SARA
Smad
2/3
Smad2/3
Smad2/3
Smad2/3
P
P
Cytoplasm
Nucleus
P
Smad4
Smad4
Smad4
Smad4
Smurf1/2
Smad3
P
Smad4Smad2/3
TFP
SBE TGF-specific miRNA genesTGF-specific genesSBE
TFSmad2/3
Smad4
P
miRNA genes
pri-miRNA
pre-miRNA
miRNAs
p68
p68
p72
miRNA processing
p72
AAAA
DGCR8
DGCR8
Drosha
Drosha
m7G
TRENDS in Pharmacological Sciences
Figure 1.
Outlineof thecrosstalkbetweenmembers of thecanonical TGF-b signalingand themiRNAmachinery. TheTGF-b/activinbindsto its
receptor. Through ligandbinding
the type II receptors phosphorylate (hence activate) type I receptors. This complexthen intracellularly activates SMADmolecules (R-SMADs) byphosphorylation. Bindingof R-
SMADs to thetype I receptoris mediated by theprotein namedSARA(SMADanchor for
receptor activation). SMAD2andSMAD3are
TGF-b-specific, whereas SMAD1, SMAD5
andSMAD8areBMP-,AMH- andGDF-specific. After activation
byTGF-b,
SMAD2/3 interactswithSMAD4and this complex translocates to thenucleus.Here
SMADcomplex can
interact withother cofactors and transcription factors, and binds to specific DNAsequences, referred to as SBE (SMAD-binding element), in promoters of TGF-b targetgenes.
Among SMAD family members, SMAD6 and SMAD7
have inhibitory roles
(I-Smad). SMAD6 preferentially inhibits phosphorylation of SMAD1/5/8 via BMP type I receptor,
whereas SMAD7 in a complex withSmurf1/2 (E3ubiquitinligases) translocates fromnucleus andassociates with TGF-b/activin type I receptor causing its degradation.SMAD7
can inhibit both the TGF-b
andBMPsignaling pathways [1,2,10].
TGF-b target
genes includegenes encoding both proteins andmiRNAs (left
lower part of the figure). Red and
green arrows indicate the connections between TGF-b signaling and biogenesis of miRNAs. In the nucleus, SMAD3 can interact with
Drosha, a member of miRNA
microprocessing complex, andcan enhance processing of boththe T/BmiRNAs(see details in thetext) andmiRNAs transcribed fromnon-T/BmiRNAgenes (right lower part of
the figure). After the pre-miRNAmature miRNA process, mature miRNA can target
severalmembers of TGF-b
signaling (highlightedas red
combs).
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Canonical miRNA biogenesis
miRNA
miRNAnmiRNA2
Transcription
Transcription
Non-canonical steps
RNA Pol II
miRNA1
Individual miRNA gene
miRNA genes in cluster
pri-miRNA
pri-miRNA
pre-miRNA
pre-miRNA
pre-miRNA
ac-pre-miRNA
miRISC
3UTR
Ribosome5
AGO2
AAAA
A
Mature miRNAmiRNA:miRNA* duplexmiRNA*
Host gene mRNACytoplasm
Nucleus
Splicing
Exon1
Exon1 Exon2
Exon2Exon1
miRNA
miRtron
Exon2
Exportin 5
Ran GTP
DGCR8
Drosha
Splicosome
Microprocessor
complex
Processing
RNA Pol II
RNA Pol II
Cleavage
Incorporationto miRISC
Cleavage
Passenger strand degradation
Translational repression,mRNA degradation by cleavage or deadenylation
Targeting mRNA
AGO2
m7G
m7G
m7G
AAAA
AAAA
AAAA
TRBP
Dicer
TRENDS in Pharmacological Sciences
Figure 2.
Biogenesis and function of miRNAs. Genes encoding miRNAs can be located in the genome individually or in clusters (upper part of the figure) of noncoding
sequences, or in introns of protein-coding genes (called miRtrons) [74]. miRNA clusters are transcribed together. Both individual and clustered miRNA genes are
transcribed by RNA polymerase II. They have a 7-methyl guanosine cap and are polyadenylated similar to mRNA molecules [117119]. The primary transcript (primary
miRNA, pri-miRNA) is processedby an RNase III (Drosha) containing complex [120]. Pri-miRNAs areprocessed by an RNase III (Drosha)containing complex.Droshacleaves
both strands into a 6070 nucleotide precursor-miRNA (pre-miRNA), which has hairpin secondary structure. The microprocessor complex contains an RNA-binding
protein (DGCR8or Pasha) and other components including DEAD boxhelicases p68and p72 [120].
Thepre-miRNA molecule is transported to the cytoplasmby Exportin-5
[121] and processed by another RNase III enzyme (Dicer) complexed with transactivation-responsive RNA binding protein (TRBP). Dicer cleaves pre-miRNA into 21 nt
miRNA:miRNA* duplexes[122]. One strand of thisRNAduplex (guide strand or maturedmiRNA) is incorporated intomiRNA-induced silencing complex (miRISC),whereas
the other strand (passenger strand ormiRNA*) is usuallydegraded[123].
However,recentdatasuggestthat miRNA* could alsobe loaded intomiRISC,which has functional
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Biogenesis and function of miRNAs
miRNAs are approximately 1925 nucleotides
long
(with
an
average of
22 nucleotides),
noncoding RNA
molecules
that post-transcriptionally regulate gene expression via
RNA
interference
by binding either
to
the 30UTR or
50UTR or the coding sequence of
protein-encoding
mRNAs [1216] (Figure 2). Approximately 3050% of
all protein-coding genes
might be controlled by miRNAs[17,18]. One miRNA has the potential to affect the ex-
pression
of
several
proteins,
and one protein
is influenced
by several
miRNAs. As the expression
pattern of the
miRome is highly tissue-specific, miRNAs provide fine-
tuning of
protein
expression
level
that
renders
them
cell-
and context-specific regulators of the adaptation process
[19]. As
miRNAs may influence many different
mRNAs,
they
can participate in the regulation
of
several
physio-
logical and pathological cellular processes. Their roles
have already been
considered
in development,
cell pro-
liferation, differentiation,
apoptosis
and
tumorigenesis
[20,21].
Experimentally
validated
interactions
between
TGF-b
signaling and miRNAs suggest that miRNAs influence theTGF-b
pathway
at
multiple
levels.
In
addition,
TGF-b
signaling itself enhances the maturation of miRNAs
[22], resulting
in
a
bidirectional
functional
link.
Regulation of the TGF-b signaling by miRNAs via direct
interaction with downstream members of canonical
signaling pathways
Most, if not all, members of the canonical TGF-b signaling
pathway
may
be
influenced
by
miRNAs.
Using in silico
miRNAmRNA
target
predictions,
several
possible
inter-
actions can be obtained. However, these predictions always
need
to
be
confirmed
experimentally.
Owing
to
the
lack
of
high-throughput
screening
(HTS)
methods
for
monitoringmiRNAmRNA
interactions,
few
such
interactions
have
been demonstrated to date (Table 1). Starting from TGF-b
receptors, it
has
been
shown
that
TGF-b
type
1
receptor
(TGF-bR1)
and
SMAD2
were
upregulated
in
most
primary
anaplastic thyroid carcinoma-derived cells, whereas miR-
NAs
(miR-30
and/or
miR-200
families)
potentially
target-
ing
these
molecules
were
downregulated
[23]. Inhibition
of
the TGF-bR1 in these cells induced EMT and a concomi-
tant
increase
of
the
miR-200
family,
suggesting
their
role
in TGF-b-mediated EMT.
In acute promyelocytic leukemia (APL) cells, miR-146a
was downregulated by all-trans-retinoid acid treatment
during
APL
differentiation.
This
miRNA
may
possibly
influence
cell
proliferation
in this
cell
line
via
SMAD4
[24].
In addition to tumorigenesis, TGF-b signaling also plays
an
important
role
in
the
development
of
several
organs.
Regulation
of
the
TGF-bR1 by
let-7
may
modulate
and
control
TGF-b
signaling
activity
to
the
necessary
level
at
each
developmental
stage
[25]. The
miR-23b
cluster
(in-
cluding miR-23b, miR-27b and miR-24-1) was found to
repress
bile
duct
gene
expression
in
fetal
hepatocytes
through
downregulation
of
SMADs
(SMAD3,
SMAD4
and SMAD5), but low levels of the miR-23b cluster was
required
in
cholangiocytes
to
allow
TGF-b
signalingnecessary for bile duct formation [26].
TGF-b
has
also
been
implicated
in
regulation
of
fibro-
genesis.
In
the
heart,
downregulation
of
miR-133
and
miR-
590 via TGF-b1 and TGF-bR2 contribute to the enhance-
ment
of TGF-b
signaling
[27], whereas
in
the
liver
and
lung, miR-21 targeting the negative regulator SMAD7 can
also
enhance
TGF-b
signaling
[28,29]. Structural
remodel-
ing in
vascular
smooth
muscle
cell
(VSMC)
can
lead
to
atherosclerosis or abdominal aortic aneurysm. In this
process,
miR-26a
was
found
to
be
a
potential
pathogenic
factorby
altering
TGF-b
signaling
through
direct
targeting
of SMAD1 [30]. miR-141 and miR-200a directly inhibit
TGF-b2
in
rat
proximal
tubular
epithelial
cells
(NRK52E),
and their downregulation may be responsible for the de-velopment
and
progression
of
TGF-b-dependent
EMT
and
fibrosis [31].
SMAD3
has
also
been
found
to
be
a
potential
miRNA
target
in
stem
cells
and
in
the
pituitary.
Interestingly,
five
of seven miRNAs that negatively correlated with tumor
size
in
pituitary
adenomas
have
been
potentially
predicted
to
target
SMAD3,
and
among
them
miR-140
was
already
validated experimentally [11,32].
Regulation of TGF-b signaling by miRNAs that interact
directly with TGF-b target genes
TGF-b
target
genes
can
also
be
regulated
by miRNAs
(Figure 3a). The
miR-106b/25
cluster
(miR-106b,
miR-93and
miR-25)
was
found
to
be
upregulated
and
correlated
with the loss of tumor suppressor activity of TGF-b signal-
ing.
These
miRNAs
directly
target
the
cell
cycle
inhibitor
p21Waf1/Cip1 and
the
pro-apoptotic
protein
BCL2L11
(BIM)
in gastric cancer by interfering with TGF-b-induced cell
cycle
arrest
and
TGF-b-mediated
apoptosis
[5].
The
miR-
106b/25
cluster
accumulates
prostate
and
pancreatic
can-
cers, neuroendocrine tumors, neuroblastoma and multiple
myeloma.
In
B
cells,
BIM
expression
is
also
affected
by
a
well-characterized oncogenic miRNA cluster, miR-17/92
[33]. This cluster impairs TGF-b effects not only by target-
ing individual TGF-b responsive genes as p21Waf1/Cip1 and
BIM
but
also
by
targeting
canonical
TGF-b
signaling
molecules
(TGF-bR2,
SMAD2,
SMAD4)
[34,35].
As discussed above, miRNAs regulate EMT by targeting
ZEB1,
ZEB2
(see
also
below),
SNAIL1,
SNAIL2
and
consequences in certain cases [124,125]. This mechanism is called canonical miRNA processing. There are two noncanonical steps in themiRNAmaturization process.
Some pre-miRNAs are transcribed from very short introns (called miRtrons) as a result of splicing anddebranching [126].
In the cytoplasmsome of these pre-miRNAs are
cleaved by AGO2, an argonaute protein into AGO2-cleaved precursor miRNA (ac-pre-miRNA). The single-stranded matured miRNA directly associates with argonaute
proteins(in mammals AGO14), which are core components of miRISC. HeremiR interacts with 30UTR of its targetmRNA by base-pairingand represses expression of the
targets. In this process theseed regionofmiRNA is essential, although there is evidence that the central loop regionof miRNA is also involvedin the target determination
[127]. The seed region is defined as the consecutive stretch of approximately seven nucleotides starting from either the first or the second nucleotide at the 50end of the
miRNA molecule. The mechanistic details of miRNA-mediated translational repression are not fully understood. Of the numerous factors that influence pairing, each
predicted miRNAmRNA target pair needs to be experimentally verified because a simple, high-throughput method for biological validation of miRNA targets does not
exist. Although several studies revealed spatial or temporal avoidance of miRNA coexpression with target genes, these may support the negative correlation between
miRNA and its potential target mRNA expression strengthening target pairing [8,22].
However, the negative correlation between mRNA and miRNA expression is not
exclusive because experimentally valid targets can be found even without changes in mRNA expression [32].
The repression of the target is realized by three major
processes: mRNA cleavage by AGO2, mRNA degradation by deadenylation and inhibition of different steps of the translation process [128132].
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HMGA2. TGF-bR2 and SNAIL2 are direct targets of miR-
204. The expression of this miRNA was significantly lower
in
the
NCI60
tumor
cell
line
panel
than
in
normal
tissues
[36]. In
addition,
SNAIL2
is
an
essential
mediator
of
EMT,
which underlines the pathogenic role of miR-204 in pro-
moting
tumor
dissemination
[37]. The
HMGAs
are
low
molecular
weight,
nonhistone
chromosomal
proteins
that
interact with the minor groove of manyAT-rich promoters
and
enhancers,
and
mutations
of
the HMGA gene associ-
ates
with
many
common
diseases,
including
benign
and
malignant tumors [38,39]. HMGA2 was found to cooperate
with
the
TGF-b
pathway
in
regulating
the
expression
of
SNAIL1
and
SNAIL2
[40,41]. There
is
a
physical
interac-
tion
between
HMGA2
and
SMAD
molecules,
leading
to
an
increased binding of SMADs to the SNAIL1 promoter [41].
Because
SNAIL1
is
a
master
downstream
effector
of
HMGA2 during induction of EMT, miRNAs may also
influence EMT by regulating HMGA2. Indeed, the fre-
quently
downregulated
miRNA
let-7
targets
HMGA2
resulting
in
its
overexpression,
which
has
already
been
demonstrated in numerous tumors, including ovarian car-
cinoma,
retinoblastoma,
uterine
leiomyosarcoma,
neuro-
endocrine
tumors
and
pituitary
adenomas
[4246].
In
these tumors, expression of HMGA proteins is associated
with
malignant
phenotypes
and
poor
prognosis.
TGF-b alters miRNA expression directly and by
regulating the maturation process of miRNAs
As
described
above,
miRNAsdirectly regulate the expres-
sion
of
members
of the canonical TGF-b signaling and
the
expression of TGF-b target genes. However, TGF-b itself
can
alter
the expression
of numerous miRNAs
through
Table
1.
Regulation
of
members
of
canonical
TGF-b
signaling
by
miRNAs
Name of miRNA miRNA target Tissue/cell where this miRNAmRNA regulation was demonstrated Species Refs
miR-133 TGF-b1 Atrial fibroblast cells Dog [27]
miR-744 TGF-b1 Proximal tubular epithelial cells (HK-2) Human [133]
miR-200a TGF-b2 NRK52E kidney proximal tubular epithelial cells Rat [31]
miR-141 TGF-b2 NRK52E kidney proximal tubular epithelial cells Rat [31]
miR-210 AcvR1B (ALK4) ST2 bone marrow-derived stromal cells Human [60]
miR-21 TGF-b components
(TGF-bR2, TGF-bR3;DAXX, BMPR2)
U251and U87glioblastoma cells Human [58]
let-7 TGF-bR1 Adult and 912 week human embryonic liver tissues Human [25]
miR-200 family,
miR-141
TGF-bR1 Anaplastic thyroid carcinoma tissues and ATC-derived cells,
proximal tubular epithelial cells (NRK52E)
Human, rat [23,134]
miR-106b,
miR-93
TGF-bR2 SH-SY5Y neuroblastoma cells, embryonic fibroblasts (MEF) Human, mouse [135,136]
miR-17-5p,
miR-20
TGF-bR2 HCT116 and DLD1 colon carcinoma cell line Human [81]
miR-204 TGF-bR2 hfRPE human fetal retinal pigment epithelial cells Human [36]
miR-20a TGF-bR2 Lung cancer tissues Human [137]
miR-21 TGF-bR2 Myometrial smooth muscle cells, leiomyoma smooth muscle cells,
transformed LSMC and SKLM-S1 leiomyosarcoma cell line
Human [57]
miR-21 TGF-bR2 hADSC human adipose tissues derived stem cell Human [56]
miR-590 TGF-bR2 Atrial fibroblast Human [27]
miR-155 SMAD1 Burkitts lymphoma cell line (Mutu I) Human [73]
miR-26 SMAD1 HeLa S3 Human [138]
miR-26a SMAD1 hADSC human adipose tissues derived stem cell Human [139]
miR-199a SMAD1 Pluripotent C3H10T1/2 stem cells Human [140]
miR-26a SMAD1
(SMAD2/3/4 reporter)
Vascular smooth muscle cells Human [30]
miR-141,
miR-200a,c,
miR-30d,e
SMAD2 Anaplastic thyroid carcinoma tissues and ATC-derived cells Human [23]
miR-155 SMAD2 THP1 monocyte cell line Human [70]
miR-140 SMAD3 Pluripotent C3H10T1/2 stem cells Human [32]
miR-23b cluster
(miR-23b, -27b, -24)
SMAD3, -4, -5 HBC-3 fetal mouse liver stem cell Mouse [26]
miR-146a SMAD4 NB4 cell (acute promyelocytic leukemia cell) and dermal fibroblast Human [25,141]
miR-146b-5p SMAD4 Papillary carcinoma cell lines (TPC-1 and BCPAP) Human [142]miR-18 SMAD4 HCT116 and DLD1 colon carcinoma cell line Human [63]
miR-224 SMAD4 Preantral granulosa cells (GCs) Mouse [138]
miR-130a SMAD4 HEK-293 kidney, A549 lung, 32Del3 myeloid precursor cell line Human [143]
miR-124 SMAD5 HeLa S3 Human [144]
miR-155 SMAD5 Diffuse large B cell lymphoma and Burkitts lymphoma
cell line (Mutu I)
Human
[71,72]
miR-21 SMAD7 Lungs of mice with bleomycin-induced fibrosis and
lungs of patients with idiopathic pulmonary fibrosis
Human,
mouse [29]
miR-21 SMAD7 HCV-infected human liver tissues Human [28]
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binding of
p68, a
component of
the Drosha
microproces-
sor
complex.
These issues
are
discussed in the following
sections.
Changes in miRNA expression levels after TGF-b
treatment
Upon
TGF-b
treatment,
changes
in
the
expression
of
nu-
merous
miRNAs
have
been
detected
in
different
cells
(Table 2).
The
miR-200
family
(miR-200a,
miR-200b,
miR-200c,
miR-141
and
miR-429)
and
miR-205
were
markedly
down-regulated
in
response
to
TGF-b
treatment
in
kidney
(MDCK) and rat proximal tubular epithelial cells
(NRK52E)
[31]. These
miRNAs
regulate
the
expression
of the
E-cadherin
transcriptional
repressors,
ZEB1
and
ZEB2, which are implicated in EMT and tumor metastasis
[47,48].
In
murine
mammary
epithelial
cells
(NMuMG),
expression
of
the
miR-200
family
was
lost
after
induction
of
EMT by TGF-b stimulation [47]. Enforced expression of the
miR-200
family
alone
was
sufficient
to
prevent
TGF-b-
induced EMT, and its inhibition was sufficient to induce
EMT in a process requiring upregulation of ZEB1 and/or
ZEB2 [49]. However, miR-200a and miR-141 directly tar-
get TGF-b
and
TGF-bR1
in
mesenchymal
anaplastic
thy-
roid
carcinoma-derived
and
NRK52E
cells
[23,31]. ZEB1
directly suppresses transcription of miR-141 and miR-
200c, and
triggers
a
miRNA-mediated
feed-forward
loop,
which
stabilizes
EMT
and
promotes
invasion
of
cancer
cells
[50]. This network, with the miR-200 family in the center,
contributes
to
the
regulation
of
EMT
in an
environmental-
ly-dependent
manner
[48].
TGF-b signaling can also regulate the expression of a
subset
of
miRNAs
via
transcription
regulation
by
SMAD3.
miRNA
let-7d
was
repressed
in tissue
samples
of
patients
with
idiopathic
pulmonary
fibrosis
(IPF)
[51], and
miR-24
was also repressed in C2C12 cells leading to reduced
expression
of
myogenic
differentiation
markers
[52].
An onco-miRNA,
miR-21
was
found
to
be
upregulated
by
TGF-b
treatment
in
breast
cancer
and
proliferating
tubu-
lar epithelial cells (TECs) [5355], and its direct interac-
tion with
TGF-bR2
was
also
demonstrated
[56,57].
Downregulation
of
miR-21
in
glioblastoma
cells
caused
growth repression, increased apoptosis and cell cycle arrest
[58]. miR-21
was
also
among
those
miRNAs
(miR-21,
miR-
32, miR-137,
miR-346,
miR-136,
miR-192,
miR-210,
miR-
211), which were suggested to participate in the regulation
of EMT
[59].
In addition
to
its
role
in
tumorigenesis,
upregulation
ofmiR-21
was
also
detected
in
myofibroblasts
obtained
both
from lungs of mice having bleomycin-induced fibrosis and
patients
with
IPF
[29].
Another
miR,
miR-210,
was
also
found
to
be
overex-
pressed after BMP4 administration and was considered to
act
as
a
positive
regulator
of
osteoblastic
differentiation
by
inhibiting
TGF-b
signaling
through
inhibition
of
ACVR1B
(ALK4) [60].
As mentioned
above,
TGF-b1 is
a
key
mediator
of
fibro-
tic diseases. The pathomechanism of this process includes
SMAD7, a direct target of mir-21. Upregulation of miR-21
in primary pulmonary fibroblasts inhibits SMAD7, and
inhibition
of
this
inhibitory
SMAD7
results
in
enhanced
TGF-b
signaling
[28,29]. SMAD3
also
regulates
miR-192
by binding to its promoter and hence participating in the
regulation
of
renal
fibrosis
[61].
Members
of
the
miR-17/92
cluster
implicated
in
tumori-
genesis (miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1,
miR-92a-1)
were
upregulated
by
TGF-b
administration
in
several
cell
lines
(Table
2) [62]. However,
miR-17/92
di-
rectly targets TGF-bR2 and SMAD4, and thus it partici-
pates
in the
regulation
of
an
autoregulatory
feedback
loop
similar
to
that
reported
between
miR-200a
and
TGF-b/
TGF-bR1
[63]. In
this
loop,
a
zinc
finger
protein
512B
which is also known as GM632 or GAM (GAM/ZFp/
ZNF512B),
a
vertebrate-specific
zinc
finger
factor,
has
a
TGF-signaling
(a) (b)
Apoptosis EMT
HMGA2PDGFB
SNAIL
DEC1
BIM
GADD45bPDCD4
miR-106b/25miR-17/92
miR106b/25miR-17/92
miR-223miR-429
miR-183 miR-204 let-7
GAM TGFBRII
SMAD4
miR-17/92Drosha
c-MYC
TGF-signaling
TGF-downstream targets
ZEB1, ZEB2, p21, BIM
DAPKc-MYC
p15p21
Tumor suppression
TRENDS in Pharmacological Sciences
Figure 3.
Schematicrepresentation of therole of theTGF-b
pathway in tumorigenesis. (a)Maindownstream effectors of the TGF-b
signaling and its fine-tuningbymiRNAs
(detailed in the text). Green arrows indicate downregulation and red arrows showupregulation. (b) Schematic regulatory feedback loop involving TGF-b
signaling (C-MYC,
ZEB1, ZEB2, p21, BIM), Drosha, miR-17/92 cluster and GAM.
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key role. miR-17/92, together with let-7, reduces GAM
expression
by
directly
targeting
its
30UTR,
whereas
GAM
downregulates
miR-17/92
via
three
mechanisms:
(i) impairs transcriptional activation of the miR-17/92
cluster
via
C-MYC;
(ii)
decreases
the
transcriptional
activ-
ity of SMADs; and (iii) by interacting directly with Drosha
is involved in pri-miRNA (primary transcript of miRNA)
miR-17/92 processing (Figure 3b). GAM increases apopto-
sis,
reduces
cell
proliferation
and
modulates
levels
of
E2F1
and
Ras.
C-MYC
itself
is
a
TGF-b
target
gene
that
acti-
vates the promoter of this miRNA cluster. In addition, the
miR-17/92
cluster
also
targets
individual
TGF-b
respon-
sive
genes,
such
as
p21Waf1/Cip1 and
BIM
[35,64].
Expression of miR-451 was shown to be induced by
SMAD3
and
SMAD4
through
SMAD
target
sites
in
their
promoter
regions
in
glioblastoma
cells,
resulting
in
cell
growth inhibition [65].
The
miR-24
cluster
(miR-23a,
miR-27a
and
miR-24)
was
also
induced
in
response
to
TGF-b
in
human
hepatocellular
carcinoma
cells
(Huh-7)
in
a
SMAD-dependent
manner.
This cluster can function as an antiapoptotic and prolifer-
ation-promoting
factor
in
liver
cancer
cells,
because
its
expression is highly upregulated in hepatocellular carci-
noma
tissues
compared
with
normal
liver
[66].
TGF-b
induces
miR-216a
and
miR-217,
which
were
shown to activate Akt. Via this mechanism, TGF-b signal-
ing
participates
in
fibrosis,
hypertrophy
and
survival
in
glomerular mesangial cells [67]. Both miR-216a and miR-
217 target phosphatase and tensin homolog (PTEN), an
inhibitor ofAkt activation. Because PTEN protein acts as a
tumor
suppressor,
this
interaction
may
have
a
delicate
role
in carcinogenesis
beyond
the
development
of
diabetic
kid-
ney disease.
Upon TGF-b
treatment
of
normal
murine
mammary
gland
(NMuMG)
epithelial
cells,
miR-155
was
shown
to
be among the most significantly elevated miRNAs. This
induction
was
SMAD4-dependent
[68]. Inhibition
of
miR-
155 was
sufficient
to
suppress
the
TGF-b-induced
EMT,
making this miRNA a potential target in breast cancer
treatment.
Interestingly,
miR-155
was
downregulated
by
TGF-b
in
normal
human
lung
fibroblasts,
but
its
ectopic
overexpression
increased
cell
migration
[69]. In
mouse
models of lung fibrosis the expression level of miR-155
was
correlated
with
the
degree
of
fibrosis
[69]. TGF-b
Table
2.
miRNAs
regulated
by
TGF-b
administration
Name of miRNA Direction of miRNA
expression changes
Tissue/cell Species Refs
let-7d Decreased Idiopathic fibrosis pulmonary tissue Human [51]
miR-142-3p Elevated Limb primary mesenchymal cells Chicken [145]
miR-145 Decreased Mesenchymal stem cells Murine [146]
miR-145 Elevated Coronary artery smooth muscle cell Human [147]
miR-146a Elevated Langerhans cells Human [148]
miR-155 Elevated Normal mouse mammary gland epithelialcells (NMUMG)
Mouse [68]
miR-155 Decreased Lung fibroblasts Human [69]
miR-17/92 cluster Elevated HEK-293 kidney, HepG2 liver, MCF7 breast
cancer cell line
Human [62]
miR-18 Elevated HeLa (cervix epithelial adenocarcinoma) Human [149]
miR-181b Elevated Hepatocellular carcinoma, breast cancer Human [150,151]
miR-192 Elevated Human kidney tubular epithelial cells
Mouse mesangial cells (MMCs)
Rat proximal tubular epithelial cells (NRK52E)
Human
Mouse
Rat
[61,152,153]
miR-200a/b Decreased Gastric cancer cell line Human [154]
miR-200a/b/c Decreased Proximal tubular epithelial cells (NRK52E) Rat [31]
miR-205;
miR-200 family
Decreased Mesenchymal cells Human [49]
miR-206 Decreased C2C12 myoblasts Mouse [155]
miR-21 Elevated Breast cancer,
human proliferating tubular epithelial cells (TECs),
rat proximal tubular epithelial cells (NRK52E)
Human,
rat
[5355,134,156]
miR-216/217 Elevated Glomerular mesangial cells Human [157]
miR-216a Elevated Mouse mesangial cell (MMCs) Mouse [152]
miR-224 Elevated Ovarian granulosa cells Mouse [158]
miR-23a/27a/24 cluster Elevated Huh-7, HepG2, Hep3B liver cells Human [55]
miR-24 Decreased C2C12 myoblasts Human [52]
miR-24 Elevated for short time,
decreased for long time
treatment
HaCaT keratinocytes Human [159]
miR-24 Elevated HeLa (cervix epithelial adenocarcinoma) Human [149]
miR-27b Decreased Cardiomyocytes Mouse [160]
miR-29 Decreased Primary murine hepatic stellate cells,
immortalized murine hepatic stellate cells,C2C12 myoblasts
Mouse [161,155]
miR-34 Decreased HeLa (cervix epithelial adenocarcinoma) Human [149]
miR-451 Elevated Glioblastoma stem (CD133+) cells Human [65]
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treatment
may
cause
opposite
effects
on
the
expression
of miR-155
in
different
cells,
underlining
again
that
both
TGF-b
and
miRNA
machineries
work
in
a
cell-
and
environment-dependent manner, and emphasizing that
the
exact
mechanisms
and
the
direct
targets
of
miR-155
have
to
be
identified
in
each
cell
type.
miR-155
directly
targets TGF-b-specific SMAD2 in THP-1 monocyte cell
lines
[70]
and
BMP-specific
SMAD1
and
SMAD5
[7173], suggesting the presence of another possible feedback
regulatory
loop.
SMADS influence miRNA processing
Daviset al. presented another mechanism that contributes
to the modulation of miR expression [74,75]. TGF-b treat-
ment
resulted
in upregulation
of
pre-miRNAs
and
matured
miRNAs,
but
not
that
of
pri-miRNAs.
These
miRs
are
regulated post-transcriptionally by a genome-independent
mechanism
through
association
of
receptor-specific
SMAD
(R-SMAD),
SMAD1
and
SMAD5,
but
not
SMAD4
proteins,
with p68, an RNA helicase component of the Drosha mi-
croprocessor
complex
[75]. This
subset
of
miRNAs
is
called
TGF-b/BMP-regulated miRNAs (T/B miRNAs) [75]. T/BmiRNAs
contain
in
their
primary
transcripts
a
conserved
sequence, identified as R-SBE, which is similar to SMAD-
binding
element
(SBE)
[76]. SMAD
proteins
through
their
amino
terminus
MH1
domain
directly
associate
with
R-
SBE. Davis et al. demonstrated that mutations in the R-
SBE region
abolished
TGF-b/BMP-mediated
induction
of
pre-miRNA
synthesis
and
impaired
pri-miRNA
binding
to
Drosha and DGCR8 in vivo [76]. In the human genome, 44
T/BmiRNAs
have
been
identified
[76]. The
nucleocytoplas-
mic shuttling
of
SMADs
(controlled
by
phosphorylation
of
serine residues by the TGF-bR1) is crucial for SMAD-
mediated
miRNA
maturation.
MAPK
and
GSK-3b
can
also
alter the
subcellular
localization
of
SMADs
through
phos-phorylation
[77,78]. Therefore,
it
was
suggested
that
SMAD regulation of miRNA processing could be modulated
independently
of
TGF-b
and
BMPs
by
signals
that
alter
the
nuclear
localization
of
SMADs
(ERKMAPK
and
the
Wnt
pathways) [76].
Similar
to
SMAD
proteins,
the
RNA
helicases
p68
have
been
shown
to
interact
with
several
other
transcription
factors, including MyoD, Runx2, androgen receptor, estro-
gen receptor
and
p53.
Association
of
p53
and
p68
facilitated
Drosha processing of a subset of miRNAs, which were
different from T/B miRNAs [79].
Pharmacological
interventions
affecting
TGF-b
signaling
The TGF-b signaling pathway is a promising target in
cancer
therapy.
Indeed,
several
compounds
affecting
this
signaling
pathway
are
under
preclinical
development
or
even in clinical trial phase, as summarized in several
recent
reviews
[8082].
From
a
theoretical
point
of
view,
there
are
three
major
possibilities in targeting the TGF-b pathway. Ligand traps
include
TGF-b
antibodies
and
soluble
TGF-b
receptors.
In
mice,
anti-TGF-b
antibodies
suppressed
metastasis
forma-
tion [83], whereas
in
the
rat,
they
arrested
progressive
nephropathy [84]. TGF-b antibodies increase the immune
response
in
animal
experiments
[85]. Anti-TGF-b1,
-b2
and pan-TGF-b
antibodies
are
under
preclinical/clinical
trials, and
have
been
tested
for
scleroderma,
prevention
of
scarring,
metastatic
melanoma
and
renal
cell
carcinoma
[80,84,86,87]. Soluble receptors, TGF-bR2 (sTbRII) and
TGF-bR3
(sTbRIII) can
also
inhibit
TGF-b
signaling.
In
hepatoma
cells
transfected
with
sTbRII,
decreased
tumor
formation was observed in an in vivo animal model [88]. In
a
transgenic
mouse
mammary
tumor
model,
increasedapoptosis in primary tumors, reduced tumor cell motility,
reduced
intravasation
and
a
decreased
number
of lung
metastases
were
detected
after
systemic
treatment
with
sTbRII [89]. sTbRIII was also tested and proved to sup-
press
cell
growth
and
metastasis
of
human
breast
cancer
and colon carcinoma cells [90].
Silencing
of
TGF-b signaling
by
antisense
oligonucleo-
tides
is
another
possibility
for
therapy.
DNA
oligonucleo-
tides can inhibit the synthesis of TGF-b1 and -b2 by
specific
binding
to
their
mRNAs.
It
has
been
shown
that
administration
of
TGF-b
antisense
oligonucleotides
can
reactivate tumor-specific immune responses [91,92].
TGF-b
antisense
nucleotides
have
been
tested
in preclini-
cal and clinical studies for the treatment of glioma, pan-creatic,
colorectal,
prostate
and
non-small
cell
lung
cancer
[86,9399].
A
third
option
of
TGF-b-targeted
therapy
is
the
use
of
intracellularly-acting
TGF-bR1
kinase
inhibitors.
Numer-
ous compounds are under development, with promising
results;
they
are
effective
in blocking
TGF-b-induced
EMT
in
mammary
epithelial
cells,
pancreatic
carcinoma
cells
and in inhibiting glioma tumor growth, as well as suppres-
sing
renal
fibrosis
in
obstructive
nephropathy
[100103].
Because
miRNAs
influence
cellular
processes
at
several
points, they are both promising therapeutic agents and
targets
[104]. Potential
drugs
that
inhibit
overexpressed
(oncogenic)
miRNAs
or
those
that
substitute
underex-pressed
(e.g.,
tumor
suppressor)
miRNAs
would
be
useful
in cancer therapy. At present, miRNA-based therapeutic
approaches
are
in experimental,
preclinical
or
in
early
clinical
phases.
The
principal
problem
is
the
delivery
of
miRNAs to the targeted cell [105]. Treatment with miR-
NAs
appears
to
be
difficult
even
when
a
local
delivery
approach
may
be
suitable
to
ensure
the
cell-specific
effect.
Systemic delivery is more dangerous; cytotoxic effects
related
to
unconjugated
miRNAs
or
vectors
used
for
deliv-
ery, as well as immunogenic reactions, present major
difficulties. For this purpose, adenoviralvectors have been
commonly used. Their application as therapeutic agents
has
been
reported
as
local
or
targeted
treatments
(e.g.,
intratumoral
delivery
in
hepatocellular
carcinoma
[106]
and transnasal administration for lung cancer treatment
[107]). Systemic
administration
of
miR-10b
inhibited
the
metastasis
formation
in
a
mouse
mammary
tumor
model.
However, cytotoxic effects related to unconjugated miR-
NAs
or
vectors
used
for
delivery,
as
well
as
immunogenic
reactions,
present
difficulties
and
cause
serious
problems
during systemic delivery [108,109]. For miRNA silencing,
several
strategies,
including
anti-miRNA
oligonucleotides
(AMOs),
miRNA
sponges
and
miRNA
masking,
have
al-
ready
been
tested.
AMOs
are
synthetic
antisense
oligonu-
cleotides that bind their target miRNAs and thereby
competitively
inhibit
the
miRNAmRNA
interaction
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[110]. In
clinical
trials,
AMOs
have
been
tested
against
hypoxia-induced
factor
1
(HIF-1a),
miR-122
and
protein
kinase
N3
(PKN3)
in
solid
tumors
[111]
and
lymphomas
[112]. Another potential area in which miRNA therapy
may
be
considered
is
treatment
of
viral
infections.
The
first
experimental
drug
was
an
antagomir,
which
inhibits
miR-
122, essential for the accumulation of hepatitis C virus
(HCV)
in
hepatic
cells.
Subcutaneous
administration
dra-matically reduced HCV load in the liver and blood [113].
Effective
treatments
may
require
knockdown
of
multiple,
rather
than
individual,
miRNAs.
For
this
specific
aim,
miRNA sponges have been developed. These compounds
are
oligonucleotide-based
constructs
with
multiple
binding
sites against an miRNA cluster inserted invectors contain-
ing
a
strong
promoter
[114]. Also,
target
protection
or
miRNA
masking
represents
an
interesting
novel
strategy
for knockdown of the effect of miRNAs. For this purpose,
single-stranded,
chemically-modified
oligoribonucleotides,
perfectly
complementary
to
the
miRNA
binding
site
of
the
30UTR of target mRNA, have been used in vivo in a zebra-
fish model
[115].
Double-stranded RNAs, which mimic the effect of en-dogenous
miRNAs,
have
been
developed
for
miRNA
sub-
stitution [105].Application of miRNAs in combination with
other
therapeutic
agents
may
also
contribute
to
a
more
successful
treatment.
Adjuvant
miRNA
therapy
may
en-
hance the effect of other systemic therapies through influ-
encing
radiosensitivity
or
increasing
sensitivity
to
DNA-
damaging
drugs.
It
has
been
reported
that
inhibition
of
the
overexpressed miR-128a (which target TGF-b-R1) leads to
resensitization
for
the
growth
inhibitory
effects
ofTGF-b
in
letrozole-resistant
breast
cancer
[116].
Concluding remarks
The
TGF-b
signaling
pathway
is
a
complex
network
thatcontrols
many
physiological
and
pathophysiological
pro-
cesses. Its regulation and interaction with miRNAs in a
cell-
and
context-specific
manner
provides
a
fine-tuning,
dynamic
and
adaptive
control
of
protein
expression.
In
the
process of tumorigenesis, alterations of the TGF-b path-
way
either
by
genetic
or
epigenetic
events
result
in
a
switch
from
a
tumor
suppressor
to
a
tumor
promoting
effect.
Recent knowledge on targeting members of this signaling
cascade by
miRNAs
or
other
agents
may
lead
to
the
devel-
opment of novel approaches in the therapy of cancer and
other diseases.
Disclosure
statement
The
authors
have
nothing
to
disclose.
AcknowledgmentsThe authors acknowledge the financial support from the Hungarian
Ministry of National Resources (ETT40/09) and TAMOP-4.2.2.B-10/B-10/
1-2010-0013. A.P. is a recipient of the Janos Bolyai Research Fellowship.
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