Journal of Pathology

J Pathol 2011; 223: 205–218 INVITED REVIEW

Published online 18 October 2010 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/path.2785

The dynamic roles of TGF-β in cancer

Erik Meulmeester1 and Peter ten Dijke1,2 *
1 Department of Molecular Cell Biology and Centre for Biomedical Genetics, Leiden University Medical Center, Postbus 9600, 2300 RC, Leiden,
The Netherlands
2 Uppsala University and Ludwig Institute for Cancer Research, Box 595, 75124, Uppsala, Sweden

*Correspondence to: Peter ten Dijke, Department of Molecular Cell Biology and Centre for Biomedical Genetics, Leiden University Medical Center,
Building 2, Room R-02-022, Postzone S-1-P, Postbus 9600, 2300 RC, Leiden, The Netherlands e-mail: p.ten_dijke@lumc.nl

Abstract
The transforming growth factor-β (TGF-β) signalling pathway plays a critical and dual role in the progression of
human cancer. During the early phase of tumour progression, TGF-β acts as a tumour suppressor, exemplified
by deletions or mutations in the core components of the TGF-β signalling pathway. On the contrary, TGF-β also
promotes processes that support tumour progression such as tumour cell invasion, dissemination, and immune
evasion. Consequently, the functional outcome of the TGF-β response is strongly context-dependent including
cell, tissue, and cancer type. In this review, we describe the molecular signalling pathways employed by TGF-β
in cancer and how these, when perturbed, may lead to the development of cancer. Concomitantly with our
increased appreciation of the molecular mechanisms that govern TGF-β signalling, the potential to therapeutically
target specific oncogenic sub-arms of the TGF-β pathway increases. Indeed, clinical trials with systemic TGF-β
signalling inhibitors for treatment of cancer patients have been initiated. However, considering the important
role of TGF-β in cardiovascular and many other tissues, careful screening of patients is warranted to minimize
unwanted on-target side effects.
Copyright  2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Keywords: BMP; cancer; epithelial to mesenchymal transition; EMT; metastasis; signal transduction; Smad; TGF-β; ubiquitin

Received 3 August 2010; Revised 18 August 2010; Accepted 1 September 2010

No conflicts of interest were declared.

Introduction pre-clinical models [6,7]. Nonetheless, issues with dos-

Transforming growth factor-β (TGF-β) emerged with
the evolution of multi-cellular organisms, where it
plays an essential role in the development of the
body plan during embryogenesis and is crucial for tis-
sue homeostasis. The TGF-β pathway mediates such
regulation through control of proliferation, differentia-
tion, apoptosis, adhesion, invasion, and cellular micro-
environment [1–4]. Consequently, malfunctioning of
this pathway has adverse effects and inactivation of
components in this signalling pathway is central to
many diseases including the development of tumouri-
genesis and tumour progression. In several types of
human carcinomas, mutations or loss of heterozy-
gosity (LOH) in central components of the TGF-β
pathway has been observed [5]. These observations
support the idea that the TGF-β pathway has a tumour-
suppressive role that needs circumvention to allow
tumourigenesis. Also, due to its growth-suppressive
effects, in the past TGF-β itself has been regarded
as an attractive cytokine for the treatment of can-
cer. Therefore, studies were initiated in which TGF-β
was explored as an adjuvant for chemotherapy. Indeed,
TGF-β was able to protect normal cells and sensitize
tumour cells towards standard chemotherapy in some
Copyright  2010 Pathological Society of Great Britain and Ireland.
Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
ing and timing of TGF-β treatment, among others,
halted the translation of these findings towards clinical
application.
Importantly, analysis of human tumour samples
has also suggested an active role for the TGF-β
pathway in tumour progression. Immuno-staining for
TGF-β correlated with metastasis in breast, colon,
and prostate cancer [8–10]. In addition, the intensity
of TGF-β staining in invading lymph node metas-
tases was higher in breast and colon cancers than
in the primary tumour [11,12]. Moreover, TGF-β
increases the motility and invasion of certain can-
cer cells, demonstrating that these cells, while hav-
ing lost sensitivity for TGF-β-induced growth arrest,
remained TGF-β-responsive [13]. Furthermore, TGF-
β secreted by tumour cells stimulates stroma forma-
tion and immune evasion of tumour cells [14,15].
Thus, TGF-β has both tumour-suppressive and tumour-
promoting functions [16]. In this review, we will
elaborate on the dynamic role that TGF-β plays in
cancer biology; how perturbation of the TGF-β sig-
nalling pathways contributes to cancer; and how new
insights provide opportunities for targeted therapy of
cancer patients.
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206
Molecular mechanism of TGF-β signalling
Canonical signalling via Smad proteins
TGF-β is the founding member of a larger family
of secreted dimeric cytokines that comprise TGF-
βs, activins, bone morphogenetic proteins (BMPs),
and growth and differentiation factors (GDFs) [4]. In
mammals, three alternative TGF-β isoforms exist, ie
TGF-β1, TGF-β2, and TGF-β3. The bioactive ligands
are composed of homo- or hetero-dimers of polypep-
tides that are synthesized as precursor molecules and
matured by proteolytic cleavage by endoproteases such
as Furin [17–19]. The cleaved N-terminal peptide, also
known as latency-associated peptide (LAP), forms a
non-covalent interaction with the C-terminal peptide
dimer (TGF-β). Subsequent binding to the latent TGF-
β-binding protein (LTBP) allows the formation of the
large latency complex (LLC) that facilitates secretion
into the extracellular matrix [20]. By release from this
complex, TGF-β is activated.
Active TGF-β dimers mediate signalling through the
TGF-β type I and type II receptors (TGF-βRI and
TGF-βRII, respectively) that are endowed with ser-
ine/threonine kinase activity [21–23]. The membrane-
anchored proteoglycan betaglycan (TGF-βRIII) assists
TGF-β binding to TGF-βRII [24]. High affinity bind-
ing of TGF-β to TGF-βRII leads to heterotetrameric
complex formation with TGF-βRI, which results in the
phosphorylation of TGF-βRI by TGF-βRII (Figure 1).
In most cell types, TGF-βRI (also known as activin
receptor-like kinase 5; ALK5) transduces signalling,
while in certain cell types ALK1 or other type I recep-
tors can mediate signalling responses [25,26]. The type
I receptor propagates signalling by recruitment and
phosphorylation of receptor-regulated Smad (R-Smad)
proteins. Whereas ALK5 signalling is mediated by
phosphorylation of Smad2 and Smad3 proteins, ALK1
signalling is mediated by Smad1, Smad5, and Smad8.
Activated Smads form a complex with the common
Smad (co-Smad; Smad4 in mammals) and shuttle into
the nucleus [21,27]. Since activated Smad complexes
have a weak binding affinity for DNA, additional
DNA-binding transcription factors are required to reg-
ulate high affinity interaction and specificity [28,29].
A variety of transcription factor families have been
described to act in concert with Smad proteins such
as p300/CBP, Forkhead, homeobox, zinc-finger, AP1,
Ets, and basic helix-loop-helix families [30,31]. The
diversity of R-Smad/co-Smad/co-factor combinations
regulates the transcription of a vast amount of target
genes. The cell type-specific responses observed upon
TGF-β stimulation can at least in part be explained by
the differential expression of these regulators in such
cell types [32].
Regulation of TGF-β Smad signalling
To control the intensity and duration of TGF-β sig-
nalling, each step in this signalling pathway is under
Copyright  2010 Pathological Society of Great Britain and Ireland.
Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
E Meulmeester and P ten Dijke
intense regulation [33]. Presentation and sequestration
of R-Smads to TGF-β receptor is controlled by SARA
and TMEPAI, respectively [34,35]. Phosphorylation
of Smad proteins is reversed by phosphatases (eg
PPM1A, PDP, and SCP1, 2 and 3), thereby creating
a rapid activation–deactivation cycle [36–38]. Fur-
thermore, activated Smad2/3 proteins are counteracted
by ubiquitin E3 ligases that target them for proteaso-
mal degradation [39,40]. In addition, the interaction
between phosphorylated Smad2 and Smad4 is impeded
by mono-ubiquitination of Smad4. Ectodermin is a
ubiquitin E3 ligase that targets Smad4 for mono-
ubiquitination, which is counteracted by the deubiqui-
tinating enzyme USP9x [41]. Moreover, the inhibitory
Smads (I-Smads), Smad6 and Smad7, are transcription-
ally induced upon BMP and TGF-β signalling [42–45].
While Smad6 mainly inhibits BMP signalling, Smad7
has the capability to prevent both BMP and TGF-β
signalling. Mechanistically, it has been proposed that
I-Smads repress signalling by competing with R-Smads
for receptor binding [43,44,46]. Secondly, it was shown
that overexpression of Smad6 prevents the formation of
Smad1–Smad4 complexes, by binding to Smad1 [47].
Smad7 has been implicated in the recruitment of the
E3 ubiquitin ligases Smurf1 and Smurf2 to activated
TGF-β receptors, which in turn leads to their degra-
dation via the ubiquitin proteasome system [48–50].
The importance of Smad7-mediated inhibition of TGF-
β signalling is underscored by the observation that
Smad7 is overexpressed in endometrial carcinomas and
thyroid follicular cell lines [51,52]. Another point of
regulation of TGF-β signalling is by the transcriptional
repressors Ski, SnoN, and members of the HDAC fam-
ily that interact with Smad2/3 and Smad4 [53–56].
Even though Smad signalling is required for the
majority of TGF-β-mediated signalling, not all re-
sponses to TGF-β are solely dependent on Smad4
[57,58]. For example, transcriptional intermediary fac-
tor 1γ (TIF1γ) selectively binds receptor-phosphory-
lated Smad2/3 in competition with Smad4, in the
control of haematopoietic cell fate by TGF-β [59]. In
addition, IKKα was identified as a critical co-regulator
of Smad2/3 in a Smad4-independent manner, which
controls keratinocyte differentiation [60].
Non-canonical signalling
While canonical signalling directly regulates the tran-
scription of Smad-dependent target genes, Smad
proteins have also been shown to participate in seques-
tration, recruitment, and enzyme activation [61–63].
R-Smads (independent of Smad4) are also involved in
the regulation of miRNA maturation in the nucleus
[64]. Moreover, alternative signalling modules are
present in parallel with Smad signalling that are
also responsive to TGF-β. The existence of Smad-
independent signalling is supported by the identifi-
cation of the TRAF6–TAK1–p38/JNK pathway as
a TGF-β signalling module downstream of TGF-β
receptors [65–67]. Furthermore, TGF-β signalling is
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TGF-β in cancer 207

Figure 1. The TGF-β signalling pathway. Transforming growth factor-β (TGF-β) present in the extracellular matrix resides in the large
latent complex (LLC). Upon release, active TGF-β weakly interacts with the large betaglycan membrane protein that is present in vast
excess compared with TGF-βRII. Subsequently, TGF-β is presented to TGF-βRII, which leads to the formation of a heterotetrameric
complex between the serine/threonine kinases TGF-βRI and TGF-βRII. The constitutive active TGF-βRII phosphorylates (P) TGF-βRI, which
in turn recruits, phosphorylates, and activates Smad transcription factors (Smad2/3). Phosphorylated Smad2/3 form a complex with the
co-Smad (Smad4), translocate into the nucleus, and further build transcription complexes with additional co-repressors or co-activators to
regulate the expression of a wide variety of genes. The inhibitory Smad (Smad7) reduces further signalling by preventing phosphorylation
of Smad2/3. Besides Smad-mediated signalling, TGF-β also activates several other signalling cascades such as TRAF6–TAK1–p38/JNK,
RhoA–Rhock1, and Par6.

engaged in RhoA–Rock1 signalling that is required Mechanisms of TGF-β signalling in tumour

for the epithelial-to-mesenchymal transition (EMT)
[68,69]. The TGF-β receptor also activates Erk-MAP
kinase signalling through direct phosphorylation of Shc
on tyrosine and serine residues by TGF-βRI [70]. TGF-
βRII can also signal independently of TGF-βRI by
direct phosphorylation of, for example, Par6 that con-
tributes to EMT by a loss of tight junctions [71]. While
an increasing number of proteins have been identified
to interact with the TGF-β receptors [72,73], novel
mechanisms of Smad-independent signalling will most
likely be uncovered in the near future.
Copyright  2010 Pathological Society of Great Britain and Ireland.
Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
suppression
In several cancers, deletion or mutations of TGF-β sig-
nalling components alleviate the tumour-suppressive
effects of this pathway. In colon cancer with microsatel-
lite instability, TGF-βRII is subjected to accumulation
of replication errors, leading to inactivation of the
receptor and thus the TGF-β pathway [74]. Similarly,
mutations in TGF-βRII have been described in gastric
tumours, gliomas, and colorectal cancer [75,76]. While
mutations in TGF-βRI are less frequent, these have
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208 E Meulmeester and P ten Dijke

been observed in pancreas, ovarian, and breast can-
cer [77–79]. Downstream of the receptors, inactivating
mutations or deletions of SMAD4 are present in half
of the pancreatic tumours and to a lesser extent in gas-
trointestinal tumours [75,78]. However, tissue-specific
inactivation of Smad4 or TGF-βRII alone in mouse
models rarely leads to spontaneous tumour forma-
tion, suggesting that the outcome of interference with
TGF-β signalling is strongly dependent on the context
of individual tumours [80–85]. In fact, the tumour-
suppressive role of TGF-β is most apparent under
conditions of oncogenic stress. For example, inactiva-
tion of Smad4 or TGF-βRII in adenomatosis polyposis
coli (APC)-deficient mice potentiates the progression
of intestinal polyps to carcinoma [83,86]. Similarly,
mammary tumours initiated by polyoma virus middle-
T oncogene and premalignant lesions initiated by the
KRAS oncoprotein in skin, pancreas, and oral and
oesophagus epithelium progress faster when TGF-β
signalling is crippled [80–82,84].
Mechanistically, TGF-β has several operating arms
to achieve its tumour-suppressive effect which include
regulation of cell proliferation, apoptosis, and indi-
rectly through the tumour stroma.
Regulation of cell proliferation and apoptosis
In epithelial, neuronal, and haematopoietic cells, TGF-
β limits cell proliferation through a coordinated pro-
gramme of cytostatic gene responses (Figure 2A).
At the core of these events is the induction of
cyclin-dependent kinase (CDK) inhibitors such as
p15 (INK4B) and p21(WAF1), which are driven by
Smad3/Smad4 complexes in concert with FoxO and
Sp1 transcription factors [87–91]. p15 inhibits cell
cycle progression at the late G1 phase by interact-
ing with CDK4/6 and preventing their interaction with
cyclin D [92]. Consequently, the CDK inhibitor p27
is relocated from cyclin D–CDK4 complexes to inter-
act with and inhibit cyclin E–CDK2 complexes. p21
also inhibits the activity of cyclin E–CDK2 complexes
[4,92]. The inactivity of these CDK complexes pre-
vents phosphorylation of pRb, a major switch in cell
cycle progression, and thus progression through G1
into S phase. While the induction of p21 and p15
is important for the cytostatic effect in neuronal and
epithelial cells, in haematopoietic cells TGF-β medi-
ates its growth inhibition through the CDK inhibitor
p57 [93].
Simultaneously with the activation of cell cycle pro-
gression inhibitors, TGF-β represses the c-Myc onco-
gene that promotes cell proliferation. c-Myc is a tran-
scription factor that activates or represses transcription
depending on its target genes. Since c-Myc inhibits
transcriptional activation of p15 and p21, through Miz-
1, TGF-β has a firm grip on the regulation of these
target genes [94,95]. Furthermore, TGF-β represses the
expression of Id1, Id2, and Id3, which are nuclear
factors implicated in differentiation and progression
through the G1–S cell cycle transition [96]. More
recently, TGF-β’s anti-proliferative effect was shown
to be dependent on the eukaryotic translation initiation
factor-4F (eIF4F) [97]. Thus, TGF-β orchestrates its

Figure 2. TGF-β as a tumour suppressor. (A) TGF-β controls cell cycle progression by repressing the oncogenic Myc transcription factor,
which in turn prevents transcriptional activation of the CDK inhibitors p21 and p15 via interaction with MIZ1. TGF-β also directly
transcriptionally activates p21 and p15. p15 abolishes the interaction of cyclin D (Cyc D) with the CDK4/6 complex, thereby inactivating
the CDK. p21 (and also p27) inactivates the cyclin E (Cyc E) CDK2 complex, which leads to stalling of the cell cycle at the G1–S boundary.
(B) TGF-β inhibits apoptosis in a variety of cell types; however, the exact molecular mechanisms remain to be determined. (C) Through
non-cell autonomous signalling, TGF-β controls tumour progression via inhibition of paracrine growth factors in the tumour stroma during
the early stages of tumour development. The absence of TGF-β signalling in tumour stroma cells relieves the growth inhibitory effect on
epithelial cells, which consequently obtain increased growth, migratory, and invasive properties.

Copyright  2010 Pathological Society of Great Britain and Ireland. J Pathol 2011; 223: 205–218
Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com
TGF-β in cancer
growth-suppressive effects via functionally redundant
mechanisms, ie by slowing down cell growth progres-
sion and simultaneously de-accelerating proliferative
actions.
In addition to the regulatory role of TGF-β in cell
cycle progression, tumour progression is also limited
through control of the apoptotic response. TGF-β has
been reported to induce apoptosis in a variety of cell
types under physiological circumstances; however, the
molecular mechanisms remain ill defined (Figure 2B).
Candidates that contribute to the apoptotic functions
of TGF-β include the death receptor FAS, growth
arrest and DNA damage inducible 45β (GADD45β),
BIM, and death-associated kinase (DAPK) [98–101].
Confirmation of the physiological relevance of these
candidates awaits experimental proof using in vivo
model systems. A further thorough understanding of
the mechanisms by which TGF-β exerts its cytostatic
and apoptotic responses is important to identify key
points where tumours may hijack the TGF-β system to
favour tumour progression.
Indirect regulation of tumour suppression
In addition to cell autonomous regulation of apoptosis
and growth arrest, TGF-β further restricts growth and
tumour progression of epithelial cells through blockage
of paracrine factor production in the tumour stroma
(Figure 2C). While the impact of the stromal fibrob-
last on tumour progression has been known since
early studies on breast cancer, the role of TGF-β in
this process emerged from mouse models in which
TGF-β signalling was impaired in stromal fibroblasts
[102,103]. Overexpression of a kinase inactive mutant
of TGF-βRII in the mammary stromal cells results
in epithelial hyperplasia and increased production of
hepatocyte growth factor (HGF) [103]. These observa-
tions were strengthened by a mouse model in which
TGF-βRII was conditionally inactivated in fibroblasts,
which results in the development of squamous cell car-
cinoma of the forestomach and prostatic intraepithelial
neoplasia [104]. The elevated expression of HGF in
the TGF-βRII-deficient fibroblast led to the activation
of its receptor (Met) in adjacent epithelial cells. Fur-
thermore, the loss of TGF-βRII in fibroblasts results
in increased TGF-α and macrophage-stimulating pro-
tein (MSP), which all together contribute to augmented
tumour growth, motility, and invasion [105]. Moreover,
in an Eµ-Myc transgenic mouse lymphoma model,
TGF-β secreted from non-neoplastic macrophages
prevents tumour development by promoting cellular
senescence [106].
Tumour-promoting roles of TGF-β signalling
During the early stages of tumour development, the
TGF-β pathway operates as a tumour suppressor,
thereby preventing tumour growth. As seen in large
Copyright  2010 Pathological Society of Great Britain and Ireland.
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209
subsets of colon, pancreatic, and gastric cancers, muta-
tion or deletion of TGF-β receptors or Smads in
the TGF-β pathway leads to its inactivation or per-
turbation of signalling responses [74–76,107–110].
Tumours that acquire the ability to bypass the tumour-
suppressive arms may exploit certain aspects of TGF-β
signalling to actively promote tumour cell progression.
In fact, aggressive tumours resistant to the tumour-
suppressive effects of TGF-β preserve the core compo-
nents of TGF-β signalling. Different types of tumours
such as gliomas and breast and prostate cancer seem to
acquire mutations preferentially not in the core compo-
nents of TGF-β signalling [109,111–114]. Such human
tumours likely obtain resistance to TGF-β-mediated
growth arrest and importantly retain the ability to
exploit TGF-β signalling to induce pathways that pro-
mote EMT, tumour invasion, metastatic dissemination,
and evasion of the immune system. Therefore, tumours
with such a signature are highly aggressive.
Epithelial-to-mesenchymal transition (EMT)
and invasion
EMT is a key process during embryonic development
that contributes to the formation of the body plan and
allows differentiation of multiple tissues and organs.
Furthermore, it plays an important role during wound
healing, where keratinocytes recapitulate part of the
EMT process to acquire a more migratory character
to seal the wounded area. Importantly, EMT also
plays a pivotal role in pathological disorders such
as fibrosis and cancer progression. Even though the
EMT process is well accepted in a variety of cancer
cell models, the criticism on its relevance for human
cancer progression has only recently dwindled, due
to convincing morphological evidence of EMT at
the invasive fronts of human tumours [115–117]. To
invade normal tissue and spread to distant organs,
carcinoma cells need to lose cell polarity, cell–cell
contacts, and acquire fibroblastic-like properties. In
this process of EMT, cells become highly motile
and invasive, which allows survival in an anchorage-
independent environment and provides them with stem
cell-like properties (Figure 3). A molecular hallmark
for cells undergoing EMT is decreased expression
of epithelial cell–cell junction proteins, such as E-
cadherin and zona occludens (ZO-1), while at the same
time acquiring the expression of mesenchymal markers,
such as vimentin, α-smooth muscle actin (α-SMA), and
fibronectin [1,118–121]. Furthermore, up-regulation
of matrix metalloproteases and N-cadherin led to
the degradation of extracellular matrix proteins and
rendered tumour cells more migratory, respectively.
The identification of TGF-β as a major inducer of
EMT came from studies in cell culture. Treatment
of normal mouse breast epithelial cells with TGF-β
changes the cuboidal shape to an elongated spindle,
accompanied by a decrease in epithelial markers and
increased expression of mesenchymal markers [122].
Canonical TGF-β–Smad signalling plays a pivotal role
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210 E Meulmeester and P ten Dijke

Figure 3. TGF-β as a tumour promoter. (A) TGF-β stimulates the progression of carcinoma in situ towards a more aggressive, motile, and
invasive carcinoma. The tumour cells (yellow) acquire the capacity to invade adjacent tissues and intravasate into the blood stream.
(B) TGF-β promotes metastatic dissemination of primary breast carcinoma towards bone by up-regulating PTHrP and IL11, which in turn
activate osteoblasts (green) to secrete RANKL. RANKL leads to the differentiation of precursor cells (blue) into osteoclasts (orange), which
absorb the bone mass and release stored TGF-β. As such, a feed-forward loop is created in which TGF-β promotes the growth of bone
metastasis. (C) By inducing ANGPTL4, TGF-β primes tumour cells for dissemination towards the lungs. ANGPTL4 enhances extravasation
by dissociating vascular endothelial cell–cell junctions. TGF-β, released by the tumour cells, allows evasion of the immune response by
inactivating CD4+ and CD8+ T-cells that normally inhibit the growth of tumour cells.

in this process, as depletion of Smad3/Smad4 or over-
expression of Smad7 completely abolishes induction
of EMT [123–125]. Additionally, increased expres-
sion of Smad3 and Smad4 in the presence of con-
stitutive active TGF-βRI enhances induction of EMT
[126]. Mechanistically, TGF-β orchestrates a diverse
transcriptional network that involves transcriptional
activation of Snail, ZEB, and the BHLH family of
proteins [119]. On the other hand, TGF-β mediates
down-regulation of the microRNA-200 family that is
required for epithelial cells to undergo EMT [127].
The expression of these factors is regulated by both
Smad-dependent and Smad-independent mechanisms
that lead to the repression of epithelial cell markers
and induction of mesenchymal genes.
Shortly after the identification of TGF-β as a reg-
ulator of EMT in cell culture conditions, work from
mouse models confirmed TGF-β as a critical regulator
of EMT in vivo. During normal craniofacial develop-
ment, TGF-β3 is required for the fusion of the two
Copyright  2010 Pathological Society of Great Britain and Ireland.
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palatal shelves, a process that is dependent on EMT.
Consequently, knockout mice for TGF-β3 are con-
fronted with a cleft palate [128,129]. Furthermore,
TGF-β2 knockouts suffer from a wide variety of devel-
opmental defects that can at least in part be explained
by a dysfunctional EMT process [130]. Additionally,
in a mouse model for skin carcinogenesis, increased
expression of TGF-β1 in keratinocytes enhances EMT
and increases the rate and aggressiveness of these
tumours, induced by chemical carcinogenesis [131]. In
this model, keratinocyte-specific depletion of Smad2
stimulates EMT by promoting Smad3/Smad4-mediated
Snail transcription, indicating that Smad2 and Smad3
have differential functions [132]. Evidence for TGF-
β as a regulator of EMT in human cancer has been
suggested by Shipitsin et al, who describe an acti-
vated TGF-β pathway in isolated CD44-positive can-
cerous cells compared with normal tissue [133]. It
appears that during the TGF-β-induced EMT process,
epithelial cells also acquire stem cell-like properties
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TGF-β in cancer
besides obtaining a mesenchymal phenotype [134].
Interestingly, Ikushima et al recently reported mech-
anistic insights into how TGF-β maintains stemness in
glioma initiating cells [135]. TGF-β directly regulates
the expression of Sox4, which in turn mediates the
induction of Sox2 expression [135].
Metastatic dissemination
After invasion into adjacent tissue, the metastatic
process continues through intravasation, dissemination
to distal capillary beds, extravasation, and growth in a
distal organ [136,137]. Particular tumour types have a
tendency to metastasize to certain organs; for example,
breast tumours tend to metastasize towards the brain,
lungs, bones, and liver. This distribution pattern seems
to be dependent on the expression of a specific set of
genes rather than vasculature, blood flow, and number
of cells delivered to the receiving organ [138–140].
The role of TGF-β in the metastatic process became
evident with the observation that TGF-β immuno-
staining was much stronger in metastasized breast
tumours than in the primary tumour [11]. In addition,
expression of TGF-βRII has a negative correlation with
overall survival in oestrogen receptor (ER)-negative
breast cancer patients [141]. Importantly, chemother-
apy or radiation treatment in the MMTV/PyVmT trans-
genic model of breast metastasis led to increased TGF-
β1 levels as well as increased circulating tumour cells
and lung metastasis [142]. Administration of neutraliz-
ing TGF-β antibodies prevents this increase in metasta-
sis, thus suggesting an important role for TGF-β in the
metastatic process [142]. However, the role of TGF-β
in metastasis progression seems to be strongly context-
dependent. For example, expression of an activated
TGF-βRI transgene increases metastasis to the lungs,
while targeted deletion of TGF-βRII resulted in a sim-
ilar observation [80,143,144]. The picture of TGF-β
as a promoter of metastasis is even further obscured
by the observation that short-term stimulation with
TGF-β stimulates metastasis formation, while persis-
tent stimulation decreases the metastatic spread to the
lungs [145]. From this study, it has been proposed that
TGF-β signalling initially needs to be high in order
to acquire invasive properties for dissemination, while
upon extravasation TGF-β signalling is low to allow
proliferation at the secondary site.
Insights into the mechanism by which TGF-β stim-
ulates metastatic dissemination came from studies on
bone and lung metastasis. Primary breast and prostate
tumours have in common that they often metastasize
towards bone, a process in which TGF-β plays an
important role upon the arrival of tumour cells. The
presence of metastatic tumour cells in the bone micro-
environment leads to activation of osteoclasts, which
degrade the bone matrix and consequently release
stored TGF-β and other growth factors. TGF-β in
turn stimulates the metastatic cells to release oste-
olytic cytokines such as, amongst others, parathyroid
Copyright  2010 Pathological Society of Great Britain and Ireland.
Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
211
hormone-related protein (PTHrP) [146,147]. The post-
translationally increased levels of PTHrP subsequently
stimulate production of RANK ligand (RANKL) in
osteoblasts, which in turn promotes the differenti-
ation of osteoclast precursors and bone resorption
(Figure 3B) [148,149]. Furthermore, it was recently
demonstrated that TGF-βRII also directly phosphory-
lates the PTH receptor and via this route could also
contribute to bone resorption [72].
Transcriptome comparison, of adrenal with bone
metastasis, originated from a breast cancer epithelial
cell line inoculated into mice, led to the identification
of CTGF and IL-11 within a larger gene signature as
critical factors for bone metastasis [138]. The TGF-
β-mediated induction of IL-11 and CTGF, as well
as the acquired metastatic capacity, requires Smad4-
dependent signalling [123,150]. Besides Smad4, Smad3
is required for the induction of key target genes
in metastasis [151]. Importantly, depletion of Smad2
enhanced the metastatic process of MDA-MB-231
cells, while Smad3 prevented the metastatic spread
[151].
Besides stimulating metastatic colonization, TGF-β
also has the ability to prime tumour cells for metastatic
dissemination. By employing a cell culture-derived
TGF-β gene response signature, angiopoietin-like 4
(ANGPTL4) was identified as a key player to prime
breast cancer cells for metastasis towards the lungs
[152]. ANGPTL4 assists the tumour cells to disrupt
the lung capillaries and thereby enables pulmonary
metastasis (Figure 3C). In a previous study ANGPLT4
has also been identified within a TGF-β-responsive
gene signature that mediates metastasis towards the
lungs [139]. The presence of dominant negative TGF-
βRI or the absence of Smad4 in ER-negative breast
cancer cells prevented their capacity to metastasize
when implanted as mammary tumours.
These studies shed light on a role for TGF-β in
the metastatic process, both in the early invasion and
intravasation stage and during the later process involv-
ing extravasation and colony formation. However, most
of these studies rely on either inoculation of cell
lines or transplantation of tumours. For future work,
it would be important to study these phenomena in a
more physiological setting employing mouse models in
which metastases arise spontaneously from the primary
tumour. In such a setting, the role of TGF-β in the var-
ious metastatic processes for different types of cancer
could be investigated employing inducible knockout or
knock-in models.
Immune evasion
In addition to regulation of EMT, invasion, and
metastatic dissemination, TGF-β also supports tumour
progression by evading the immune system [2]. The
first evidence for TGF-β in immune evasion came
from the observation that TGF-β potently inhibits
tumour-induced CD8+ cytolytic T-lymphocyte (CTL)-
mediated rejection of a murine tumour [153]. Further
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212
support for TGF-β as an immuno-suppressant came
from a transgenic mouse model expressing a dom-
inant negative form of TGF-βRII (dn-TGF-βRII) in
all T-cells [154]. The absence of TGF-β signalling in
T-lymphocytes suppressed growth and metastasis in
this mouse model when challenged with melanoma
or lymphoma cell lines. By employing differential
immuno-depletion in these mice, it was determined that
both T-helper (CD4+ ) and cytolytic T-cells (CD8+ )
were responsible for tumour resistance. In addition,
inactivation of TGF-β signalling in CTLs (overexpres-
sion of dn-TGF-βRII) in a mouse model for sponta-
neous prostate cancer delays tumour progression [155].
Mechanistically, it was demonstrated that TGF-β tran-
scriptionally represses the production of several pro-
apoptotic factors in CTLs such as perforin, granzyme
A, granzyme B, FAS ligand, and interferon-γ [156].
In addition to its immuno-suppressive effect on CTLs,
TGF-β also represses the activity of natural killer (NK)
cells. Inhibition of TGF-β using neutralizing TGF-β
antibodies increased NK cell activity and consequently
resulted in the suppression of metastasis formation
of an inoculated breast carcinoma cell line [157].
CD4+ CD25+ regulatory T-cells that produce high
amounts of TGF-β take part in the inhibition of NK cell
activity [158]. TGF-β inhibits NK-mediated cytotoxic-
ity through transcriptional repression of the activating
receptor NKG2D and the type I transmembrane pro-
tein NKp30 [159,160]. Neutralizing antibodies towards
TGF-β could restore the NKG2D expression, as well
as the anti-tumour reactivity [161]. In glioblastoma
patients, TGF-β decreases the expression of NKG2D
in CTLs and NK cells, and represses the expression of
MICA (the ligand of NKG2D) in glioma cells [162].
Therapeutic exploitment of the TGF-β pathway
A rationale for designing therapeutic inhibitors that
block TGF-β signalling in human cancer was initiated
by the observation that excess TGF-β promotes tumour
progression. The current strategies to target TGF-β
mainly focus on general inhibition of TGF-β signalling,
which can be subdivided into three major approaches.
Firstly, the synthesis of TGF-β can be prevented by
administration of antisense molecules. Secondly, ligand
traps (including monoclonal TGF-β neutralizing anti-
bodies and soluble TGF-βRII or TGF-βRIII) prevent
soluble TGF-β from activating its signalling cascade.
Thirdly, small molecule inhibitors hinder the kinase
activity of TGF-β receptors.
Targeting TGF-β signalling using antisense
molecules
During tumour progression, the production of TGF-β
is often enhanced either by fibroblasts in the stroma or
by the tumour itself, which correlates with increased
aggressiveness of the tumour. One strategy to diminish
Copyright  2010 Pathological Society of Great Britain and Ireland.
Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
E Meulmeester and P ten Dijke
the production of the ectopic TGF-β is by using anti-
sense molecules, which block TGF-β-mediated gene
expression. Antisense molecules for TGF-β1 demon-
strate promising results in pre-clinical experiments
by decreasing tumourigenicity [163,164]. A promising
candidate in further development is AP 12 009 (Anti-
sense Pharma), an antisense molecule directed against
TGF-β2, which reduces TGF-β2-mediated migration
and proliferation, as well as alleviating its immuno-
suppressive effects [165,166]. The efficacy in phase
I/II clinical trials to treat refractory high-grade glioma
patients exceeded the expected median survival com-
pared with chemotherapy [166]. These results suggest
AP 12 009 as a promising therapeutic target to inhibit
TGF-β signalling for malignant tumour therapy. Cur-
rently, AP 12 009 is in further clinical studies to test
its efficacy for metastatic melanoma, pancreatic carci-
noma, and metastatic colorectal carcinoma.
Ligand traps that sequester TGF-β
Another approach to prevent TGF-β signalling is
trapping the TGF-β ligand employing soluble TGF-
β receptors or neutralizing antibodies. Soluble TGF-
βRII and TGF-βRIII have proven their value in
pre-clinical model systems. For example, expression
of soluble TGF-βRII prevents pulmonary, pancreatic
or liver metastasis in mouse models [167–169], while
intraperitoneal delivery of the soluble ectodomain of
TGF-βRIII into athymic nude mice inhibited pul-
monary metastasis [170]. Alternatively, TGF-β neutral-
izing antibodies such as 2G7 and 1D11 prevent the
interaction of TGF-β with its receptors and thereby
restrain its biological activity [157,171]. Administra-
tion of 1D11 following inoculation of 4T1 cells sup-
pressed pulmonary metastasis, while 2G7 transiently
reduced the growth rate of intra-abdominal transplanted
MDA-MB-231 cells and lung metastasis [157,172].
Based on these results, Genzyme developed a mon-
oclonal neutralizing TGF-β antibody (GC1008) for
which recently a clinical phase I/II trial was com-
pleted to assess the safety and efficacy in patients with
advanced metastatic melanoma or renal cell carcinoma.
Small molecule inhibitors of TGF-β signalling
Additionally, small molecule inhibitors that restrain the
kinase activity of TGF-β receptors can prevent TGF-β
signalling. The compound SB-431 542 prevents TGF-
β-mediated Smad2/3 phosphorylation by restraining the
kinase activity of TGF-βRI [173]. Treatment of human
osteosarcoma cells with SB-431 542 inhibits TGF-β-
induced proliferation, while in malignant glioma cells
angiogenesis, proliferation, and motility are inhibited
[174,175]. Another inhibitor of TGF-βRI is SD-208,
which upon systemic administration increases median
survival upon implantation of malignant glioma cells
into the mouse brain [176]. In another study, SD-208
was reported to inhibit TGF-β-induced EMT, migra-
tion, and invasion of two murine breast carcinoma cell
lines, while also inhibiting the metastasis formation of
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TGF-β in cancer
these cell lines when inoculated into the mammary fat
pads of mice [177]. The compound Ki26894, also a
TGF-βRI kinase inhibitor, prevents the motility and
invasion of breast and gastric cancer cells [178,179].
Furthermore, LY2 109 761 is a small molecule that
inhibits the kinase activity of both TGF-βRI and TGF-
βRII, and has been shown to inhibit the metastatic
spread to a variety of organs [180,181]. The small
molecule compound LY2 157 299 (Eli Lilly & Co) is
currently in a clinical phase I trial to address its safety
and pharmacokinetics. LY2 157 299 has been reported
in mouse studies to inhibit the primary tumour growth
of a breast and lung cancer cell line [182].
Conclusion and future perspectives
With the continuously increasing appreciation of the
clinical importance of TGF-β as a tumour promoter,
interest in targeting the TGF-β pathway for therapeutic
intervention is rising. The use of genetic screens that
delineate the tumour-suppressive versus the tumour-
promoting roles of TGF-β signalling will provide a
basis for new research that will enable the target-
ing of its specific oncogenic sub-arms. Several pre-
clinical studies led to clinical trials that underline the
potential to restrain TGF-β signalling [183,184]. Fur-
thermore, treatment using soluble TGF-βRII prevents
the metastatic spread in a mouse model, while severe
pathological disorders observed as in TGF-β knockout
mice remain absent [168]. However, some concerns
regarding the general systemic inhibition of TGF-β
remain, due to its tumour-suppressive role [74–79].
Furthermore, TGF-β also has a profound role in the car-
diovascular system, such that general TGF-β inhibitors
will most likely have adverse on-target side effects.
Therefore, to determine the treatment regimen, it is of
critical importance to define and select those patients
who are confronted with tumours in which TGF-β’s
tumour-promoting role prevails. Although individual-
based medicine still has a long way to go, the current
state-of-the-art techniques such as microarray, pro-
tein microarray or super-SILAC pave the way for a
detailed view of key corrupted pathways in individuals
[185–187].
Acknowledgment
We are grateful to all laboratory members for stimulat-
ing discussions. We apologize to those whose work
we could not cite due to limited space constraints.
Our work is supported by grants from the LUMC
(‘vrije beleidsruimte’), the Dutch Cancer Society, the
Netherlands Organization for Health and Development,
the Swedish Cancer Foundation, and the Centre for
Biomedical Genetics.
Author contribution statement
EM wrote the manuscript under the supervision of PtD.
Copyright  2010 Pathological Society of Great Britain and Ireland.
Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
213
Teaching materials
PowerPoint slides of the figures from this review
are supplied as supporting information in the online
version of this article.
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