Review

Crosstalk between TGF-b signaling and

the microRNA machinery
Henriett Butz1, Karoly Racz1, Laszlo Hunyady2 and Attila Patocs3,4
1

2nd Department of Medicine, Faculty of Medicine, Semmelweis University, Budapest, Hungary

Department of Physiology, Semmelweis University, Budapest, Hungary
3
Molecular Medicine Research Group, Hungarian Academy of Sciences, Budapest, Hungary
4
Department of Laboratory Medicine, Semmelweis University, Budapest, Hungary
2

The activin/transforming growth factor-b (TGF-b) pathway 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 regulation 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 addition, the biogenesis of miRNAs is also regulated by TGFb 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 between 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 conserved polypeptides, which play prominent roles in the
regulation of embryonic development, reproduction and
tumor 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, colorectal, 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 this
cleavage 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 disulfide 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
Corresponding author: Patocs, A. (patatt@bel2.sote.hu).
Keywords: TGF-b signaling pathway; miRNA; endocrine neoplasm.

382

complex is formed, type II receptors activate type I receptors by phosphorylation, and type I receptors subsequently
phosphorylate downstream SMAD proteins which transmit 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 membrane-anchored proteoglycan that can bind TGF-bs and
facilitates interaction of TGF-b-type II receptor with TGFb [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 the
TGF-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 oncogenic factors such as c-Myc, a transcription factor that promotes 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 signaling has tumor promoting downstream targets. For
example, through induction of deleted in esophageal cancer 1 (DEC1), platelet-derived growth factor beta polypeptide (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 epithelialmesenchymal transition (EMT). EMT is a biological process through which a polarized cell that normally
interacts with a basement membrane (epithelial phenotype) switches to a mesenchymal phenotype that is characterized by invasiveness and increased cell mobility [6].

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

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TGF-/activin

TGFBR II

Smad
2/3

TGFBR I

SARA

Smad
2/3
miRNAs

Smad4

miRNA processing

Smurf1/2
Smad4
Smad
2/3

Smad3

sm

Nucleus

p68

Cytopla

Smad4
Smad
P
2/3

p72

pre-miRNA

DGCR8
Drosha
p68

p72

DGCR8
Drosha

Smad4

m7G

AAAA

pri-miRNA

Smad4
Smad TF
2/3

SBE

Smad4 TF
P Smad2/3

TGF-specific genes

SBE

TGF-specific miRNA genes

miRNA genes
TRENDS in Pharmacological Sciences

Figure 1. Outline of the crosstalk between members of the canonical TGF-b signaling and the miRNA machinery. The TGF-b/activin binds to its receptor. Through ligand binding
the type II receptors phosphorylate (hence activate) type I receptors. This complex then intracellularly activates SMAD molecules (R-SMADs) by phosphorylation. Binding of RSMADs to the type I receptor is mediated by the protein named SARA (SMAD anchor for receptor activation). SMAD2 and SMAD3 are TGF-b-specific, whereas SMAD1, SMAD5
and SMAD8 are BMP-, AMH- and GDF-specific. After activation by TGF-b, SMAD2/3 interacts with SMAD4 and this complex translocates to the nucleus. Here SMAD complex can
interact with other cofactors and transcription factors, and binds to specific DNA sequences, referred to as SBE (SMAD-binding element), in promoters of TGF-b target genes.
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 with Smurf1/2 (E3 ubiquitin ligases) translocates from nucleus and associates with TGF-b/activin type I receptor causing its degradation. SMAD7
can inhibit both the TGF-b and BMP signaling pathways [1,2,10]. TGF-b target genes include genes encoding both proteins and miRNAs (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, and can enhance processing of both the T/B miRNAs (see details in the text) and miRNAs transcribed from non-T/B miRNA genes (right lower part of
the figure). After the pre-miRNA mature miRNA process, mature miRNA can target several members of TGF-b signaling (highlighted as red combs).

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 tumorigenesis and, in certain situations, it switches from a
tumor suppressor to a tumor promoter [2]. For the tumor

promoting effect, additional secondary molecular mechanisms, including mutation of the TP53 gene encoding tumor
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 the major mediators
of environment-dependent TGF-b-induced responses, as a
downstream collector of multiple signaling pathways, including 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 miRNAs has also been demonstrated in different tumors
[10,11].
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Canonical miRNA biogenesis

RNA Pol II

Non-canonical steps

RNA Pol II

Individual miRNA gene

RNA Pol II

miRtron

Exon1

miRNA

miRNA

Exon2

miRNA genes in cluster

miRNA1

miRNA2

miRNAn

Transcription

Transcription
Exon1

Exon

m7 G

AAAA
pri-miRNA

m7G

AAAA

pri-miRNA

DGCR8
Drosha

Nucleus

Splicosome

Processing

Splicing

Microprocessor
complex

m7 G

Exon1

Exon2

AAAA

Host gene mRNA

Cytoplasm

pre-miRNA

Ran GTP
Exportin 5

AGO2
Cleavage

pre-miRNA
pre-miRNA
ac-pre-miRNA

TRBP
Dicer

Cleavage

miRISC

Incorporation
to miRISC

Passenger strand degradation

miRNA*

miRNA:miRNA* duplex

Mature miRNA

Targeting mRNA

AAAA

Ribosome

AGO2
3 UTR

Translational repression,
mRNA degradation by cleavage or deadenylation
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 processed by an RNase III (Drosha) containing complex [120]. Pri-miRNAs are processed by an RNase III (Drosha) containing complex. Drosha cleaves
both strands into a 6070 nucleotide precursor-miRNA (pre-miRNA), which has hairpin secondary structure. The microprocessor complex contains an RNA-binding
protein (DGCR8 or Pasha) and other components including DEAD box helicases p68 and p72 [120]. The pre-miRNA molecule is transported to the cytoplasm by 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 this RNA duplex (guide strand or matured miRNA) is incorporated into miRNA-induced silencing complex (miRISC), whereas
the other strand (passenger strand or miRNA*) is usually degraded [123]. However, recent data suggest that miRNA* could also be loaded into miRISC, which has functional

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Review
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 30 UTR or
50 UTR 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 expression of several proteins, and one protein is influenced
by several miRNAs. As the expression pattern of the
miRome is highly tissue-specific, miRNAs provide finetuning of protein expression level that renders them celland context-specific regulators of the adaptation process
[19]. As miRNAs may influence many different mRNAs,
they can participate in the regulation of several physiological and pathological cellular processes. Their roles
have already been considered in development, cell proliferation, differentiation, apoptosis and tumorigenesis
[20,21].
Experimentally validated interactions between TGF-b
signaling and miRNAs suggest that miRNAs influence the
TGF-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 interactions can be obtained. However, these predictions always
need to be confirmed experimentally. Owing to the lack of
high-throughput screening (HTS) methods for monitoring
miRNAmRNA 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 miRNAs (miR-30 and/or miR-200 families) potentially targeting these molecules were downregulated [23]. Inhibition of
the TGF-bR1 in these cells induced EMT and a concomitant 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.

Trends in Pharmacological Sciences July 2012, Vol. 33, No. 7

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 (including 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 signaling
necessary for bile duct formation [26].
TGF-b has also been implicated in regulation of fibrogenesis. In the heart, downregulation of miR-133 and miR590 via TGF-b1 and TGF-bR2 contribute to the enhancement 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 remodeling 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
factor by 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 development 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-93
and miR-25) was found to be upregulated and correlated
with the loss of tumor suppressor activity of TGF-b signaling. 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 miR106b/25 cluster accumulates prostate and pancreatic cancers, 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 targeting 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 the miRNA maturization process.
Some pre-miRNAs are transcribed from very short introns (called miRtrons) as a result of splicing and debranching [126]. In the cytoplasm some 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. Here miR interacts with 30 UTR of its target mRNA by base-pairing and represses expression of the
targets. In this process the seed region of miRNA is essential, although there is evidence that the central loop region of miRNA is also involved in 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 50 end 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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Table 1. Regulation of members of canonical TGF-b signaling by miRNAs

Name of miRNA
miR-133
miR-744
miR-200a
miR-141
miR-210
miR-21

let-7
miR-200 family,
miR-141
miR-106b,
miR-93
miR-17-5p,
miR-20
miR-204
miR-20a
miR-21
miR-21
miR-590
miR-155
miR-26
miR-26a
miR-199a
miR-26a
miR-141,
miR-200a,c,
miR-30d,e
miR-155
miR-140
miR-23b cluster
(miR-23b, -27b, -24)
miR-146a
miR-146b-5p
miR-18
miR-224
miR-130a
miR-124
miR-155

miRNA target
TGF-b1
TGF-b1
TGF-b2
TGF-b2
AcvR1B (ALK4)
TGF-b components
(TGF-bR2, TGF-bR3;
DAXX, BMPR2)
TGF-bR1
TGF-bR1

Tissue/cell where this miRNAmRNA regulation was demonstrated

Atrial fibroblast cells
Proximal tubular epithelial cells (HK-2)
NRK52E kidney proximal tubular epithelial cells
NRK52E kidney proximal tubular epithelial cells
ST2 bone marrow-derived stromal cells
U251and U87glioblastoma cells

Species
Dog
Human
Rat
Rat
Human
Human

Refs
[27]
[133]
[31]
[31]
[60]
[58]

Human
Human, rat

[25]
[23,134]

TGF-bR2

Adult and 912 week human embryonic liver tissues

Anaplastic thyroid carcinoma tissues and ATC-derived cells,
proximal tubular epithelial cells (NRK52E)
SH-SY5Y neuroblastoma cells, embryonic fibroblasts (MEF)

Human, mouse

[135,136]

TGF-bR2

HCT116 and DLD1 colon carcinoma cell line

Human

[81]

TGF-bR2
TGF-bR2
TGF-bR2

hfRPE human fetal retinal pigment epithelial cells

Lung cancer tissues
Myometrial smooth muscle cells, leiomyoma smooth muscle cells,
transformed LSMC and SKLM-S1 leiomyosarcoma cell line
hADSC human adipose tissues derived stem cell
Atrial fibroblast
Burkitts lymphoma cell line (Mutu I)
HeLa S3
hADSC human adipose tissues derived stem cell
Pluripotent C3H10T1/2 stem cells
Vascular smooth muscle cells

Human
Human
Human

[36]
[137]
[57]

Human
Human
Human
Human
Human
Human
Human

[56]
[27]
[73]
[138]
[139]
[140]
[30]

Anaplastic thyroid carcinoma tissues and ATC-derived cells

Human

[23]

SMAD2
SMAD3
SMAD3, -4, -5

THP1 monocyte cell line

Pluripotent C3H10T1/2 stem cells
HBC-3 fetal mouse liver stem cell

Human
Human
Mouse

[70]
[32]
[26]

SMAD4
SMAD4
SMAD4
SMAD4
SMAD4
SMAD5
SMAD5

NB4 cell (acute promyelocytic leukemia cell) and dermal fibroblast

Papillary carcinoma cell lines (TPC-1 and BCPAP)
HCT116 and DLD1 colon carcinoma cell line
Preantral granulosa cells (GCs)
HEK-293 kidney, A549 lung, 32Del3 myeloid precursor cell line
HeLa S3
Diffuse large B cell lymphoma and Burkitts lymphoma
cell line (Mutu I)
Lungs of mice with bleomycin-induced fibrosis and
lungs of patients with idiopathic pulmonary fibrosis
HCV-infected human liver tissues

Human
Human
Human
Mouse
Human
Human
Human

[25,141]
[142]
[63]
[138]
[143]
[144]
[71,72]

Human, mouse

[29]

Human

[28]

TGF-bR2
TGF-bR2
SMAD1
SMAD1
SMAD1
SMAD1
SMAD1
(SMAD2/3/4 reporter)
SMAD2

miR-21

SMAD7

miR-21

SMAD7

HMGA2. TGF-bR2 and SNAIL2 are direct targets of miR204. 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 promoting tumor dissemination [37]. The HMGAs are low
molecular weight, nonhistone chromosomal proteins that
interact with the minor groove of many AT-rich promoters
and enhancers, and mutations of the HMGA gene associates 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 interaction 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
386

HMGA2 during induction of EMT, miRNAs may also

influence EMT by regulating HMGA2. Indeed, the frequently downregulated miRNA let-7 targets HMGA2
resulting in its overexpression, which has already been
demonstrated in numerous tumors, including ovarian carcinoma, retinoblastoma, uterine leiomyosarcoma, neuroendocrine 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, miRNAs directly regulate the expression 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

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(a)

(b)

TGF-
signaling

c-MYC

Drosha

Tumor suppression
c-MYC

p21
p15

Apoptosis

GADD45b
DAPK
PDCD4
BIM

EMT
DEC1
PDGFB
HMGA2
SNAIL
GAM

miR-106b/25
miR-17/92

miR-223
miR-429

miR-183

miR106b/25
miR-17/92

miR-17/92

miR-204

let-7

TGFBRII TGF-
SMAD4 signaling

ZEB1, ZEB2, p21, BIM

TGF- downstream targets
TRENDS in Pharmacological Sciences

Figure 3. Schematic representation of the role of the TGF-b pathway in tumorigenesis. (a) Main downstream effectors of the TGF-b signaling and its fine-tuning by miRNAs
(detailed in the text). Green arrows indicate downregulation and red arrows show upregulation. (b) Schematic regulatory feedback loop involving TGF-b signaling (C-MYC,
ZEB1, ZEB2, p21, BIM), Drosha, miR-17/92 cluster and GAM.

binding of p68, a component of the Drosha microprocessor 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 numerous 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 downregulated 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-binduced 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 target TGF-b and TGF-bR1 in mesenchymal anaplastic thyroid carcinoma-derived and NRK52E cells [23,31]. ZEB1
directly suppresses transcription of miR-141 and miR200c, 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 environmentally-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 tubular epithelial cells (TECs) [5355], and its direct interaction 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, miR32, miR-137, miR-346, miR-136, miR-192, miR-210, miR211), which were suggested to participate in the regulation
of EMT [59].
In addition to its role in tumorigenesis, upregulation of
miR-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 overexpressed 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 fibrotic 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 tumorigenesis (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 directly targets TGF-bR2 and SMAD4, and thus it participates 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
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Table 2. miRNAs regulated by TGF-b administration

Name of miRNA
let-7d
miR-142-3p
miR-145
miR-145
miR-146a
miR-155

Direction of miRNA
expression changes
Decreased
Elevated
Decreased
Elevated
Elevated
Elevated

miR-155
miR-17/92 cluster

Decreased
Elevated

miR-18
miR-181b
miR-192

Elevated
Elevated
Elevated

miR-200a/b
miR-200a/b/c
miR-205;
miR-200 family
miR-206
miR-21

Decreased
Decreased
Decreased

miR-216/217
miR-216a
miR-224
miR-23a/27a/24 cluster
miR-24
miR-24

miR-24
miR-27b
miR-29

Elevated
Elevated
Elevated
Elevated
Decreased
Elevated for short time,
decreased for long time
treatment
Elevated
Decreased
Decreased

miR-34
miR-451

Decreased
Elevated

Decreased
Elevated

Tissue/cell

Species

Refs

Idiopathic fibrosis pulmonary tissue

Limb primary mesenchymal cells
Mesenchymal stem cells
Coronary artery smooth muscle cell
Langerhans cells
Normal mouse mammary gland epithelial
cells (NMUMG)
Lung fibroblasts
HEK-293 kidney, HepG2 liver, MCF7 breast
cancer cell line
HeLa (cervix epithelial adenocarcinoma)
Hepatocellular carcinoma, breast cancer
Human kidney tubular epithelial cells
Mouse mesangial cells (MMCs)
Rat proximal tubular epithelial cells (NRK52E)
Gastric cancer cell line
Proximal tubular epithelial cells (NRK52E)
Mesenchymal cells

Human
Chicken
Murine
Human
Human
Mouse

[51]
[145]
[146]
[147]
[148]
[68]

Human
Human

[69]
[62]

Human
Human
Human
Mouse
Rat
Human
Rat
Human

[149]
[150,151]
[61,152,153]

C2C12 myoblasts
Breast cancer,
human proliferating tubular epithelial cells (TECs),
rat proximal tubular epithelial cells (NRK52E)
Glomerular mesangial cells
Mouse mesangial cell (MMCs)
Ovarian granulosa cells
Huh-7, HepG2, Hep3B liver cells
C2C12 myoblasts
HaCaT keratinocytes

Mouse
Human,
rat

[155]
[5355,134,156]

Human
Mouse
Mouse
Human
Human
Human

[157]
[152]
[158]
[55]
[52]
[159]

HeLa (cervix epithelial adenocarcinoma)

Cardiomyocytes
Primary murine hepatic stellate cells,
immortalized murine hepatic stellate cells,
C2C12 myoblasts
HeLa (cervix epithelial adenocarcinoma)
Glioblastoma stem (CD133+) cells

Human
Mouse
Mouse

[149]
[160]
[161,155]

Human
Human

[149]
[65]

key role. miR-17/92, together with let-7, reduces GAM

expression by directly targeting its 30 UTR, 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 activity 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 apoptosis, reduces cell proliferation and modulates levels of E2F1
and Ras. C-MYC itself is a TGF-b target gene that activates the promoter of this miRNA cluster. In addition, the
miR-17/92 cluster also targets individual TGF-b responsive 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 proliferation-promoting factor in liver cancer cells, because its
388

[154]
[31]
[49]

expression is highly upregulated in hepatocellular carcinoma 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 signaling participates in fibrosis, hypertrophy and survival in
glomerular mesangial cells [67]. Both miR-216a and miR217 target phosphatase and tensin homolog (PTEN), an
inhibitor of Akt activation. Because PTEN protein acts as a
tumor suppressor, this interaction may have a delicate role
in carcinogenesis beyond the development of diabetic kidney 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 miR155 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

Review
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 [71
73], suggesting the presence of another possible feedback
regulatory loop.
SMADS influence miRNA processing
Davis et al. presented another mechanism that contributes
to the modulation of miR expression [74,75]. TGF-b treatment 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 microprocessor complex [75]. This subset of miRNAs is called
TGF-b/BMP-regulated miRNAs (T/B miRNAs) [75]. T/B
miRNAs contain in their primary transcripts a conserved
sequence, identified as R-SBE, which is similar to SMADbinding element (SBE) [76]. SMAD proteins through their
amino terminus MH1 domain directly associate with RSBE. Davis et al. demonstrated that mutations in the RSBE 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/B miRNAs have been identified [76]. The nucleocytoplasmic shuttling of SMADs (controlled by phosphorylation of
serine residues by the TGF-bR1) is crucial for SMADmediated miRNA maturation. MAPK and GSK-3b can also
alter the subcellular localization of SMADs through phosphorylation [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, estrogen 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 formation [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

Trends in Pharmacological Sciences July 2012, Vol. 33, No. 7

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, increased
apoptosis 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 suppress cell growth and metastasis of human breast cancer
and colon carcinoma cells [90].
Silencing of TGF-b signaling by antisense oligonucleotides is another possibility for therapy. DNA oligonucleotides 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 preclinical and clinical studies for the treatment of glioma, pancreatic, 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. Numerous 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 suppressing 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 underexpressed (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 miRNAs 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 delivery, as well as immunogenic reactions, present major
difficulties. For this purpose, adenoviral vectors 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 miRNAs 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 already been tested. AMOs are synthetic antisense oligonucleotides that bind their target miRNAs and thereby
competitively inhibit the miRNAmRNA interaction
389

Review
[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 miR122, essential for the accumulation of hepatitis C virus
(HCV) in hepatic cells. Subcutaneous administration dramatically 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 in vectors containing 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
30 UTR of target mRNA, have been used in vivo in a zebrafish model [115].
Double-stranded RNAs, which mimic the effect of endogenous miRNAs, have been developed for miRNA substitution [105]. Application of miRNAs in combination with
other therapeutic agents may also contribute to a more
successful treatment. Adjuvant miRNA therapy may enhance the effect of other systemic therapies through influencing radiosensitivity or increasing sensitivity to DNAdamaging 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 of TGF-b in
letrozole-resistant breast cancer [116].
Concluding remarks
The TGF-b signaling pathway is a complex network that
controls many physiological and pathophysiological processes. 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 pathway 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 development of novel approaches in the therapy of cancer and
other diseases.
Disclosure statement
The authors have nothing to disclose.
Acknowledgments
The authors acknowledge the financial support from the Hungarian
MOP-4.2.2.B-10/B-10/
Ministry of National Resources (ETT40/09) and TA
1-2010-0013. A.P. is a recipient of the Janos Bolyai Research Fellowship.

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