TGF-beta Rev 2019

International Journal of
Molecular Sciences
Review
TGF-β-Mediated Epithelial-Mesenchymal Transition
and Cancer Metastasis
Yang Hao, David Baker and Peter ten Dijke *
Department of Cell and Chemical Biology and Oncode Institute, Leiden University Medical Center,
Einthovenweg 20, 2300 RC Leiden, The Netherlands; Y.Hao@lumc.nl (Y.H.); D.A.Baker@lumc.nl (D.B.)
* Correspondence: p.ten_dijke@lumc.nl; Tel.: +31-71-526-9271

Received: 18 April 2019; Accepted: 24 May 2019; Published: 5 June 2019
Abstract: Transforming growth factor β (TGF-β) is a secreted cytokine that regulates cell proliferation,
migration, and the differentiation of a plethora of different cell types. Consistent with these findings,
TGF-β plays a key role in controlling embryogenic development, inflammation, and tissue repair,
as well as in maintaining adult tissue homeostasis. TGF-β elicits a broad range of context-dependent
cellular responses, and consequently, alterations in TGF-β signaling have been implicated in many
diseases, including cancer. During the early stages of tumorigenesis, TGF-β acts as a tumor suppressor
by inducing cytostasis and the apoptosis of normal and premalignant cells. However, at later stages,
when cancer cells have acquired oncogenic mutations and/or have lost tumor suppressor gene
function, cells are resistant to TGF-β-induced growth arrest, and TGF-β functions as a tumor
promotor by stimulating tumor cells to undergo the so-called epithelial-mesenchymal transition
(EMT). The latter leads to metastasis and chemotherapy resistance. TGF-β further supports cancer
growth and progression by activating tumor angiogenesis and cancer-associated fibroblasts and
enabling the tumor to evade inhibitory immune responses. In this review, we will consider the role of
TGF-β signaling in cell cycle arrest, apoptosis, EMT and cancer cell metastasis. In particular, we will
highlight recent insights into the multistep and dynamically controlled process of TGF-β-induced
EMT and the functions of miRNAs and long noncoding RNAs in this process. Finally, we will discuss
how these new mechanistic insights might be exploited to develop novel therapeutic interventions.
Keywords: EMT; lncRNA; metastasis; miRNA; SMAD; TGF-β; targeted therapy; tumor microenvironment
1. Introduction
Cancer treatments have been refined over a period spanning several thousand years, culminating in
the primary modern approaches of surgery, chemotherapy and radiation therapy [1]. In recent decades,
more targeted and personalized treatments have gained prominence. The fundamental aim of these
therapies is to specifically kill tumor cells while leaving healthy tissues intact [2]. Despite demonstrable
progress, these strategies frequently deliver relatively modest improvements in disease outcomes
owing to acquired resistance and excessive toxicity [3]. These findings indicate the need for greater
optimization of current treatments as well as the identification of alternative targets for the development
of novel cancer remedies.
Cancer is a complex disease in which tumor cell heterogeneity and the reciprocal interplay between
tumor cells and surrounding stromal cells and extracellular matrix are key determinants in tumor
progression and therapy response. Tumorigenesis shows similarity to subverted normal embryogenic
developmental processes in which communication between cells is controlled by cytokines that act
in an autocrine, paracrine or juxtacrine manner. In this light, we will provide a general overview
of the established roles of one such important developmental cancer signaling pathway, namely
the transforming growth factor-β (TGF-β), in tumorigenesis [4]. In particular, we consider how
Int. J. Mol. Sci. 2019, 20, 2767; doi:10.3390/ijms20112767 www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2019, 20, 2767 2 of 34
this essential TGF-β signaling network orchestrates the epithelial to mesenchymal transition (EMT),
the mechanism by which cancer cells lose polarity and separate from each other, adopt the characteristics
of a mesenchymal phenotype, become motile and invade distant sites. Key TGF-β-induced effectors
in this process are the transcriptional repressors of E-cadherin, e.g., SNAIL1, SNAIL2 (also termed
SLUG), ZEB1/2 and TWIST. Moreover, miRNAs and long noncoding RNAs (lncRNAs) are emerging
as potentially quantifiable biomarkers of cancer status and are potential targets for anti-metastatic
therapies [5]. In this review, we will highlight the pivotal role of these molecules in regulating TGF-β
signaling and epithelial-mesenchymal transition (EMT).
2. TGF-β and Signaling Transduction across the Plasma Membrane

TGF-β1 (hereafter termed simply TGF-β) is the prototypic member of a large family of structurally
and functionally related proteins, which includes its close relatives TGF-β2 and TGF-β3 but also
activins, nodal, inhibins, Mullerian-inhibiting substance (MIS), growth and differentiation factors
(GDF) and bone morphogenetic proteins (BMPs) [6]. TGF-β was discovered in the early 1980s as a
secreted factor that, together with TGF-β or epidermal growth factor (EGF), induced the growth of
normal rat kidney (NRK) cells in soft agar. Since then, we have gained a much deeper understanding
of this multifunctional cytokine [7]. The TGF-β gene encodes a pre-pro-precursor peptide of 390 amino
acids. The pro-precursor peptide is proteolytically processed by furin proteases into an amino-terminal
fragment and the carboxy-terminal 112 amino acids, which corresponds to the mature bioactive
TGF-β [8]. The amino-terminal part is also termed the latency associated peptide (LAP) and is
noncovalently attached to the mature TGF-β [9,10]. Latent TGF-β is activated by specific proteases
that cleave the LAP and/or by mechanical forces generated by cell surface integrins, resulting in the
release of mature, active TGF-β [11,12]. Bioactive TGF-β, which is capable of binding its cell surface
receptors, is a dimeric protein linked by disulfide bonds with an apparent molecular weight of 25 kDa.
TGF-β exerts its cellular effects via cell surface TGF-β type I and type II receptors, e.g., TβRI and
TβRII, respectively [13]. TβRI and TβRII are structurally related and consist of an extracellular domain
characterized by the presence of cysteine residues that form disulfide bonds, a single transmembrane
domain and an intracellular region harboring a conserved serine/threonine kinase domain. TGF-β
initially engages the TβRII, which drives the recruitment of TβRI and the formation of a heterotetrameric
complex composed of two TβRIIs and two TβRIs. Subsequently, the active TβRII kinase domain
phosphorylates TβRI on specific serine and threonine residues in the glycine-serine (GS) juxtamembrane
region, which leads to its activation [14]. TGF-β receptors are widely expressed in human tissues/cells
and mediate various biological phenomena, such as embryonic development, tissue homeostasis,
organogenesis, immune surveillance and tissue repair.
3. Intracellular SMAD and Non-SMAD Signaling Pathways

Genetic studies designed to delineate the critical components of the dauer and decapentaplegic
pathway in Caenorhabditis elegans and Drosophila, respectively, led to the identification of Small (Sma)
and Mothers against dpp (Mad) genes [15,16]. The mammalian homologues of the proteins encoded
by these genes, termed SMADs, were found to act as intracellular transcriptional effectors of TGF-β
family receptor signaling [17]. The SMAD family can be divided into receptor-regulated (R-) SMADs
(in vertebrates: R-SMAD1, -2, -3, -5 and -8) that interact with and become phosphorylated by activated
type I receptor kinases, the common (Co-) SMADs (in vertebrates: SMAD4) that form heteromeric
complexes with activated R-SMADs and inhibitory I-SMADs (in vertebrates: I-SMAD6/7), which
antagonize canonical SMAD signaling [18] (Figure 1).
Int. J. Mol. Sci. 2019, 20, 2767 3 of 34
Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 3 of 37
90
91 Figure 1.1. TGF-β/SMAD
Figure TGF-β/SMAD and and non-SMAD
non-SMAD signaling.
signaling. TGF-β
TGF-β elicits
elicits its
its cellular
cellular responses
responses by
by forming
forming
92 ligand-induced
ligand-induced complex formation of TGF-β type I and type II cell surface receptors (i.e.(i.e.,
complex formation of TGF-β type I and type II cell surface receptors TβRITβRI
and
93 and TβRII) that are endowed with serine/threonine kinase activity. The extracellular signal
TβRII) that are endowed with serine/threonine kinase activity. The extracellular signal is transduced is
transduced across the plasma membrane through the action of the constitutively active TβRII kinase that
94 across the plasma membrane through the action of the constitutively active TβRII kinase that
phosphorylates specific serine and threonine residues in the intracellular juxtamembrane GS domain
95 phosphorylates specific serine and threonine residues in the intracellular juxtamembrane GS domain
of TβRI. Intracellular signaling is then initiated when the activated TβRI kinase phosphorylates or
96 of TβRI. Intracellular signaling is then initiated when the activated TβRI kinase phosphorylates or
activates intracellular signal mediators. In the case of the canonical SMAD pathway, TβRI recruits and
97 activates intracellular signal mediators. In the case of the canonical SMAD pathway, TβRI recruits
phosphorylates specific R-SMADs, e.g., SMAD2 and SMAD3, which can form heteromeric complexes
98 and phosphorylates specific R-SMADs, e.g., SMAD2 and SMAD3, which can form heteromeric
with SMAD4. These transcription factor complexes then translocate into the nucleus and cooperate with
99 complexes with SMAD4. These transcription factor complexes then translocate into the nucleus and
other transcription regulators to regulate target gene expression. In the non-SMAD pathway, TGF-β
100 cooperate with other transcription regulators to regulate target gene expression. In the non-SMAD
receptors activate other pathways, including various branches of MAPK pathways, RHO-like GTPase
101 pathway, TGF-β receptors activate other pathways, including various branches of MAPK pathways,
signaling pathways and phosphatidylinositol-3-kinase (PI3K)/AKT pathways. Inhibitory signals are
102 RHO-like GTPase signaling pathways and phosphatidylinositol-3-kinase (PI3K)/AKT pathways.
indicated with inhibitory red arrows; Stimulatory signals are indicated with green arrows.
103 Inhibitory signals are indicated with inhibitory red arrows; Stimulatory signals are indicated with
104 greenR-
The arrows.
and Co-SMADs have two conserved domains, termed Mad homology (MH)1 and
MH2 domains at the amino-terminal (MH1) and carboxy-terminal (MH2) ends of the proteins.
105 The R- and Co-SMADs have two conserved domains, termed Mad homology (MH)1 and MH2
MH1 and MH2 are separated by a flexible linker region. R-SMAD phosphorylation by type I receptors
106 domains at the amino-terminal (MH1) and carboxy-terminal (MH2) ends of the proteins. MH1 and
occurs on two serine residues, which comprise the SXS sequence motif, at the C-termini. SMAD3 and
107 MH2 are separated by a flexible linker region. R-SMAD phosphorylation by type I receptors occurs
SMAD4, but not SMAD2, bind directly to the consensus 50 -CAGA-30 DNA motif. Heteromeric complex
108 on two serine residues, which comprise the SXS sequence motif, at the C-termini. SMAD3 and
formation between R-SMADs and SMAD4 is mediated by MH2 domains. Heteromeric complex
109 SMAD4, but not SMAD2, bind directly to the consensus 5′-CAGA-3′ DNA motif. Heteromeric
formation of R-SMADs and SMAD4 exposes nuclear import signals and shields nuclear export signals,
110 complex formation between R-SMADs and SMAD4 is mediated by MH2 domains. Heteromeric
resulting in their nuclear accumulation. In the nucleus, they can act as transcription factors in concert
111 complex formation of R-SMADs and SMAD4 exposes nuclear import signals and shields nuclear
with other DNA binding transcription factors, coactivators and repressors and chromatin remodeling
112 export signals, resulting in their nuclear accumulation. In the nucleus, they can act as transcription
factors, which enable diverse transcriptional responses depending upon the particular combination
113 factors in concert with other DNA binding transcription factors, coactivators and repressors and
of proteins [19]. I-SMAD7, which has a carboxy terminal region with homology to R-SMADs and
114 chromatin remodeling factors, which enable diverse transcriptional responses depending upon the
SMAD4 MH2 domains, antagonizes the activation of TGF-β receptor/SMAD signaling. SMAD7 achieves
115 particular combination of proteins [19]. I-SMAD7, which has a carboxy terminal region with
this via multiple mechanisms, including the recruitment of the E3 ubiquitin ligase SMURF2 to the
116 homology to R-SMADs and SMAD4 MH2 domains, antagonizes the activation of TGF-β
activated receptor, thereby targeting the TGF-β receptor for proteasomal and lysosomal degradation.
117 receptor/SMAD signaling. SMAD7 achieves this via multiple mechanisms, including the recruitment
SMAD7 can also attenuate signaling by recruiting phosphatases to the activated TGF-β receptor, which
118 of the E3 ubiquitin ligase SMURF2 to the activated receptor, thereby targeting the TGF-β receptor for
mediate receptor inactivation by the dephosphorylation of specific amino acid residues [20].
119 proteasomal and lysosomal degradation. SMAD7 can also attenuate signaling by recruiting
120 phosphatases to the activated TGF-β receptor, which mediate receptor inactivation by the
121 dephosphorylation of specific amino acid residues [20].
Int. J. Mol. Sci. 2019, 20, 2767 4 of 34
TGF-β can also signal via non-SMAD pathways. This process can occur directly or indirectly [21].
An example of an indirect mechanism is TGF-β/SMAD-stimulated expression of growth factors, such
as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), which thereafter initiate
non-SMAD responses. Non-SMAD signaling can also be initiated directly downstream of the TGF-β
receptor by activation of various branches of the MAP kinase (MAPK) pathway, Rho-like GTPase
signaling pathways and the phosphatidylinositol-3-kinase (PI3K)/AKT pathway [22]. For example,
activated TβRI can induce tyrosine phosphorylation of SHCA, which associates with GRB2, and recruits
a GRB2/SOS complex, thereby triggering activation of the RAS/RAF/MAP kinase signaling cascade [23].
Activated TGF-β receptor complexes also induce K63-linked polyubiquitination of TRAF4/6, leading
to the recruitment of TAK1 and triggering its activation, thus allowing TAK1 to activate JNK signaling
through MKK4 or P38 MAPK signaling via MKK3/6 [18,24,25]. At tight junctions, TβRII phosphorylates
PAR6 at serine residue 345. This phosphorylated PAR6 recruits SMURF1 to the activated TβRI-TβRII
receptor complex. The PAR6-SMURF1 complex subsequently mediates localized ubiquitination
and turnover of RHOA at cellular protrusions [26]. TGF-β can promote the interaction between
CDC42 GTPase/RAC and p21-activated kinase (PAK) 2, an interaction that is dependent on SMAD7 [27].
TGF-β receptors interact with the regulatory p85 subunit of PI3K, resulting in activation of the
PI3K/AKT pathway, which controls translational responses through mammalian target of rapamycin
(mTOR) [28]. Activation of non-SMAD signaling occurs in a context-dependent manner, and these
pathways also crosstalk with the canonical SMAD pathway.
4. Tumor Suppressive Effects of TGF-β

TGF-β can act as a potent tumor suppressor in normal and premalignant epithelial cell types
(Figure 2). TGF-β triggers G1 phase cell cycle arrest by different mechanisms in different cell
types [29]. For example, this molecule can activate the translation-inhibitory protein 4E-BP1 (regulator
of eukaryotic translation initiation factor-4F (eIF4E)) promoter activity through SMAD4, thereby
suppressing translation and cell growth and proliferation [30]. TGF-β causes late G1 cell cycle arrest
by inducing the expression of cyclin-dependent kinase (CDK) inhibitors (p15INK4b, p21WAF1 and
p27KIP1) to inhibit CDK-cyclin complexes [31]. In MCF10A and MDA-MB-231 cell lines, the tumor
suppressor p53 is a critical SMAD partner, which promotes TGF-β-induced p21 expression to block cell
cycle progression [32]. In addition, TGF-β exerts growth inhibitory effects by inhibiting the expression
of CDC25a phosphatase (which is required for CDK-cyclin activation) [33] or negatively regulating
Id proteins (helix-loop-helix (HLH) proteins, which are essential for inhibition of cell differentiation
and growth arrest) [34] in the prostate epithelial cell line HPr-1. TGF-β directly inhibits c-MYC by
binding a transcriptional repression complex containing SMAD2/3, E2F4/5, p107, and C/EBPβ to the
TGF-β inhibitory element in the proximal region and thus achieving cell cycle arrest in HaCaT, COS-1,
and Mv1Lu-tet-p15 cells and human leukemia MO-91 cells [35].
In addition to cell cycle arrest, TGF-β can induce cell apoptosis in the early phase of
tumorigenesis [29]. However, the precise mechanisms by which TGF-β induces this effect in different
cell types remain unclear. The expression of some apoptotic regulators (such as growth arrest
and DNA damage (GADD) 45, Bcl-2-like protein 11 (BIM), BCL-2 interacting killer (BIK), death
associated protein kinase (DAPK), FAS, and B-cell lymphoma-extra large (BCL-XL)) was shown to
be regulated by the TGF-β/SMAD signaling pathway [36]. For example, BIM was found to be a key
mediator of TGF-β-induced apoptosis in intestinal adenoma cells [37] and in hepatocarcinoma cells [38].
TGF-β1-associated regulatory SMAD proteins bind to the BIK (also known as NBK) promoter, which
encodes a proapoptotic sensitizer protein in B cells [39]. After TGF-β1 treatment, researchers found
that TGF-β induced SMAD-dependent binding between the proapoptotic effector BIM and BCL-XL
in gastric carcinoma cell lines [40] and a decrease in BCL-XL expression followed by activation of
the apoptosis proteins caspase-9 and caspase-3 in human hepatoma cells (HuH-7) [41]. TGF-β can
activate the TAK1-p38/JNK pathway, which has been reported to lead to apoptosis in HEK 293T
cells [25]. This molecule also promoted the expression of SMAD-dependent GADD45β in hepatocytes
Int. J. Mol. Sci. 2019, 20, 2767 5 of 34
and plays an important role in cell death by mediating delayed TGF-β-induced p38 MAP kinase
activation [42]. Here, the effects of TGF-β in proapoptotic signaling occur in a context-dependent
manner. Additionally, the TGF-β signaling pathway can be coupled to the cell death machinery through
the induction of reactive oxygen species (ROS) [43], apoptosis genes (SHIP and TIEG), modulation
of epigenetic regulators (DNMTs) [44], H3K79me3 and H2BK120me1 [45], and telomere shortening
through regulating human telomerase reverse transcriptase (hTERT) in the breast cancer MCF-7 cell
line
Int. J.[46]. These
Mol. Sci. 2019,effects of PEER
20, x FOR TGF-β in proapoptotic signaling occur in a context-dependent manner.
REVIEW 5 of 37
158
159 Figure
Figure 2. TGF-β-induced growth
2. TGF-β-induced growth inhibition
inhibition and
and apoptosis. The TGF-β/SMAD
apoptosis. The TGF-β/SMAD pathway pathway can
can arrest
arrest cells
cells
160 in
in the
the G1
G1phase
phaseofofthe
thecell
cellcycle
cyclebybymodulating
modulatingthe theexpression
expression ofof
specific genes,
specific genes,including
including induction
inductionof
161 4EBP1,
of 4EBP1,p15INK4b,
p15INK4b,p16INK4A,
p16INK4A, p21WAF1
p21WAF1 andand
p57KIP2
p57KIP2andand
repression of Idofproteins,
repression E2F, E2F,
Id proteins, c-MYC and
c-MYC
162 CDC25a
and CDC25a genes. The The
genes. TGF-β/SMAD
TGF-β/SMAD pathway can can
pathway induce cellcell
induce apoptosis by inducing
apoptosis by inducingthethe
expression
expressionof
163 proapoptotic genes, such as BCL-XL, BIM, BIK, SHIP and TIEG. The TGF-β activation
of proapoptotic genes, such as BCL-XL, BIM, BIK, SHIP and TIEG. The TGF-β activation of TAK-1 of TAK-1 occurs
164 via TRAF6
occurs via and
TRAF6the and
adaptor XIAP-mediated
the adaptor TAB/TAK-1
XIAP-mediated complex and
TAB/TAK-1 GADD45β.
complex ActivationActivation
and GADD45β. of NF-κB
165 by the TAKbypathway
of NF-κB the TAKstimulates
pathway p38/JNK
stimulatesphosphorylation, which has been
p38/JNK phosphorylation, reported
which to lead
has been to apoptosis.
reported to lead
166 Additionally, the TGF-β signaling pathway can be coupled to the cell death machinery
to apoptosis. Additionally, the TGF-β signaling pathway can be coupled to the cell death machinery through ROS,
167 autophagy activation (ATG-5/-6/-7), induction of DAPK expression, epigenetic changes
through ROS, autophagy activation (ATG-5/-6/-7), induction of DAPK expression, epigenetic changes and shortening
168 of
andtelomere length
shortening (regulating
of telomere hTERT).
length Inhibitory
(regulating hTERT).signals are indicated
Inhibitory with
signals are inhibitory
indicated with red arrows;
inhibitory
169 Stimulatory signals are indicated with green arrows. SMAD-mediated transcriptional
red arrows; Stimulatory signals are indicated with green arrows. SMAD-mediated transcriptional events are
indicated with the black arrow.
170 events are indicated with the black arrow.
The TGF-β signaling pathway can inhibit tumor growth by multiple other mechanisms, including
171 In addition to cell cycle arrest, TGF-β can induce cell apoptosis in the early phase of
activating autophagy in certain human cancer cells. For example, in human hepatocellular carcinoma
172 tumorigenesis [29]. However, the precise mechanisms by which TGF-β induces this effect in different
cell lines, TGF-β induced the accumulation of autophagosomes and increased the expression levels of
173 cell types remain unclear. The expression of some apoptotic regulators (such as growth arrest and
the autophagy markers Autophagy-related 5 (ATG5), Beclin1, ATG7 and death-associated protein kinase
174 DNA damage (GADD) 45, Bcl-2-like protein 11 (BIM), BCL-2 interacting killer (BIK), death associated
(DAPK) [47]. Moreover, siRNA-mediated silencing of autophagy genes attenuated TGF-β-mediated
175 protein kinase (DAPK), FAS, and B-cell lymphoma-extra large (BCL-XL)) was shown to be regulated
growth inhibition and induction of the proapoptotic genes BIM and BMF in human hepatocellular
176 by the TGF-β/SMAD signaling pathway [36]. For example, BIM was found to be a key mediator of
carcinoma cells [48].
177 TGF-β-induced apoptosis in intestinal adenoma cells [37] and in hepatocarcinoma cells [38]. TGF-β1-
Because of its central role in tumor suppression, TGF-β signaling components were found to
178 associated regulatory SMAD proteins bind to the BIK (also known as NBK) promoter, which encodes
be mutated and functionally inactivated in various cancers [49]. The first example was SMAD4,
179 a proapoptotic sensitizer protein in B cells [39]. After TGF-β1 treatment, researchers found that TGF-
which is frequently mutated in gastrointestinal cancers [50]. Subsequently, other TGF-β signaling
180 β induced SMAD-dependent binding between the proapoptotic effector BIM and BCL-XL in gastric
181 carcinoma cell lines [40] and a decrease in BCL-XL expression followed by activation of the apoptosis
182 proteins caspase-9 and caspase-3 in human hepatoma cells (HuH-7) [41]. TGF-β can activate the
183 TAK1-p38/JNK pathway, which has been reported to lead to apoptosis in HEK 293T cells [25]. This
184 molecule also promoted the expression of SMAD-dependent GADD45β in hepatocytes and plays an
Int. J. Mol. Sci. 2019, 20, 2767 6 of 34
components, e.g., TGF-β receptors [51] and SMADs (SMAD2 and SMAD3), were found to be mutated
in various cancers, including bladder, colon, breast, esophageal, stomach, brain, liver, and lung
cancers [52]. Loss of tumor suppressor function and epigenome and microenvironmental changes can
also affect the tumor-promoting activity of the TGF-β receptor/SMAD pathway [53]. For example,
in gastrointestinal tumors, TβR1 activity was decreased due to the methylation status of the TβR1
promoter [54]. In turn, TGF-β/SMAD can affect the epigenome of genes involved in cancer processes.
TGF-β and SMAD2/3 show oncogenic activities, such as promoting glioma cell proliferation, by affecting
the methylation status of the platelet-derived growth factor-β (PDGF-B) gene and autocrine PDGF-B
signaling within tumor microenvironments [55]. TGF-β stimulated myofibroblast percent and invasion
rate in tumor-associated fibroblasts (CAFs) that increase tumor invasion [56].
5. TGF-β-induced Tumor Promoting Effects
5.1. Cell Biology of TGF-β-induced EMT

In the late stage of cancer progression, cancer cells remain responsive to TGF-β but become resistant to
its cytostatic effects. In fact, by acting directly on cancer cells, TGF-β can promote tumorigenesis by inducing
the so-called epithelial to mesenchymal transition (EMT) [57]. Under normal physiological conditions,
EMT plays a crucial role in the context of embryogenesis and tissue damage repair [58]. This process can
be subverted and pathological, and EMT drives the development of fibrotic disease and tumorigenesis [59].
EMT is characterized by changes in the levels of three prominent biomarkers (E-cadherin, vimentin,
and N-cadherin), and these changes can lead to decreased adhesion of cells, loss of polarity and tight
junctions. At the same time, epithelial cells adopt the traits of a mesenchymal phenotype, notably motility
and susceptibility to invasion and metastasis (Figure 3) [60]. Importantly, mesenchymal cancer cells are
correlated with poor prognosis and associated with resistance to chemotherapy [61]. Interestingly, recent
studies have questioned the necessity of EMT in establishing metastasis [62]. Zheng et al. reported that
in genetically engineered mouse models of pancreatic adenocarcinoma development and spontaneous
metastasis mouse models of breast cancer, tumor cells could metastasize without activating EMT programs,
and EMT only contributed to chemoresistance [63]. Similarly, Fischer et al. also showed that EMT is not
required for lung metastasis but contributes to chemoresistance in spontaneous breast-to-lung metastasis
models [64]. However, these findings have been challenged by other researchers, who believed that Zheng
et al. failed to completely suppress the activation of EMT, and their results can only speak to the redundancy
of EMT within the transcriptional network in pancreatic carcinomas. These researchers continue to
subscribe to the notion that EMT is required for metastatic dissemination in pancreatic carcinoma cells [65].
Increasing data have shown that EMT is not a single, stereotypical program but instead has a
multistep process that passes through intermediate hybrid states (partial EMT state, P-EMT) during the
transition from epithelial to mesenchymal cells [66]. The TGF-β-induced transition from epithelial to
P-EMT is reversible, whereas the transition from P-EMT to mesenchymal cells is potentially irreversible
depending on the type of cell that is involved [59]. Studies have found that there are at least seven
tumor subpopulations associated with different EMT stages in skin and breast cancer tissues, which
contributes to intratumor heterogeneity. These EMT subpopulations displayed differences in cellular
plasticity, invasiveness, and metastatic potential, and tumor cells with an early stage of EMT were most
likely to metastasize [67]. An interesting paper reported that TGF-β activates scleroderma epithelial
cells to the P-EMT process in fibrotic skin [68]. Related to this, single cell RNA-seq showed that P-EMT
plays an important role in head and neck cancer, and further in vitro analyses suggested that TGF-β
dynamically controls the transition between P-EMT and non-P-EMT states in cells [69].
Int. J. Mol. Sci. 2019, 20, 2767 7 of 34
235
236 Figure
Figure 3.3. TGF-β mediates EMT.
TGF-β mediates EMT. TGF-β
TGF-β isis aa strong
strong promoter
promoter of of EMT,
EMT, which
which is
is characterized
characterized by
by aa loss
loss
237 of epithelial and gain of mesenchymal markers. Polar epithelial cells remodel into highly
of epithelial and gain of mesenchymal markers. Polar epithelial cells remodel into highly migratory migratory
238 mesenchymal
mesenchymal cells,
cells, followed by decreased
followed by decreased adhesion of cells
adhesion of cells and
and loss
loss of
of polarity
polarity and
and tight
tight junctions.
junctions.
239 TGF-β via SMAD or non-SMAD signaling pathways can induce the expression of
TGF-β via SMAD or non-SMAD signaling pathways can induce the expression of several EMT-TFs, several EMT-TFs,
240 such
such asas SNAIL1,
SNAIL1, SNAIL2,
SNAIL2, ZEB1/2
ZEB1/2 and
and TWIST.
TWIST. The The migratory
migratory andand invasive
invasive mesenchymal-like
mesenchymal-like tumor
tumor
241 cells
cells extravasate from primary lesions into blood or lymphatic vessels and then intravasate
extravasate from primary lesions into blood or lymphatic vessels and then intravasate to
to distant
distant
242 sites
sites via,
via, where
where they
they form
form metastatic
metastatic colonies
colonies upon
upon MET.
MET.
5.2. Molecular Mechanisms in TGF-β-induced EMT
243 Increasing data have shown that EMT is not a single, stereotypical program but instead has a
244 SMAD
multistep levels/activities
process that passes mediate
through TGF-β-induced EMT by
intermediate hybrid inducing
states theEMT
(partial expression of E-cadherin
state, P-EMT) during
245 transcriptional
the transition fromrepressors, such
epithelial as SNAIL, ZEBcells
to mesenchymal and[66].
TWIST,
The which cooperatetransition
TGF-β-induced with otherfromtranscription
epithelial
246 regulators
to P-EMT in the nucleuswhereas
is reversible, [58]. Several additional
the transition from lines of evidence
P-EMT argue forcells
to mesenchymal a roleis of SMADs
potentially
247 in TGF-β-induced
irreversible dependingEMT,on and someofexamples
the type cell that isare provided
involved [59].below. Knockdown
Studies have found thatof SMAD4
there are in
at
248 MDA-MB-231
least seven tumor breast cancer cells robustly
subpopulations attenuated
associated bone metastasis
with different EMT stagesin nude miceand
in skin andbreast
significantly
cancer
249 prolonged
tissues, whichsurvival of the treated
contributes animals [70].
to intratumor SMAD3 and
heterogeneity. SMAD4
These EMT interact to form adisplayed
subpopulations complex
250 with SNAIL1
differences that targets
in cellular the tight-junction
plasticity, invasiveness, protein (CAR) potential,
and metastatic and E-cadherin
and tumorduring
cellsTGF-β-driven
with an early
251 EMT
stage of EMT were most likely to metastasize [67]. An interesting paper reported that TGF-βinhibited
in breast epithelial cells. Conversely, co-silencing of SNAIL1 and SMAD4 by siRNA activates
252 repression
scleroderma ofepithelial
CAR andcells
occludin
to the during
P-EMT EMT
process[71]. In addition,
in fibrotic SMAD3/SMAD4-mediated
skin [68]. Related to this, single cellSNAILRNA-
253 transcription
seq showed that contributed to EMT
P-EMT plays an during skinrole
important carcinogenesis, whilecancer,
in head and neck SMAD2 loss
and significantly
further in vitroincreased
analyses
254 this effect [72].
suggested thatMoreover, SMAD7, the
TGF-β dynamically transcriptional
controls target between
the transition and negativeP-EMTregulator of TGF-β signaling,
and non-P-EMT states in
255 upregulated
cells [69]. TGF-β and inducing SMAD7 transcription prevented TGF-β-induced EMT and invasion
of cancer cells [73]. Additionally, ubiquitin ligases that promote poly-ubiquitination and proteasomal
256 5.2. MolecularofMechanisms
degradation in TGF-β-induced
SMADs affect EMT is illustrated by E3 ubiquitin ligase RNF8, which
EMT. This finding
activates TWIST via K63-linked ubiquitination to promote EMT and cancer stem cell (CSC) self-renewal,
257 SMAD levels/activities mediate TGF-β-induced EMT by inducing the expression of E-cadherin
resulting in enhanced metastasis and chemoresistance in breast cancer [74].
258 transcriptional repressors, such as SNAIL, ZEB and TWIST, which cooperate with other transcription
TGF-β can also induce EMT in a non-SMAD-dependent fashion, for example, by promoting cytoskeletal
259 regulators in the nucleus [58]. Several additional lines of evidence argue for a role of SMADs in TGF-
remodeling, which leads to activation of ERK [75]. The ERK required for cytoskeletal remodeling interacts
260 β-induced EMT, and some examples are provided below. Knockdown of SMAD4 in MDA-MB-231
with SHC or GRB2 to form an SHC-GRB2-ERK complex, which is a key component of TGF-β-induced
261 breast cancer cells robustly attenuated bone metastasis in nude mice and significantly prolonged
tumor invasion and metastasis [76]. ERK substrates, AP-1 family members, enhance SMAD transcriptional
262 survival of the treated animals [70]. SMAD3 and SMAD4 interact to form a complex with SNAIL1
263 that targets the tight-junction protein (CAR) and E-cadherin during TGF-β-driven EMT in breast
Int. J. Mol. Sci. 2019, 20, 2767 8 of 34
activity to regulate gene expression and TGF-β-induced EMT [77]. However, the RHO-like GTPases,
including RHOA, RAC and CDC42, are also involved in TGF-β-induced EMT. TGF-β regulates cytoskeletal
changes via mediating RHO GTPase to achieve the dissolution of tight junctions among cells. TGF-β
mediates the RHOA activity level and promotes the activation of LIM kinase (LIMK) by Rho-related kinase
(ROCK) and phosphorylated myosin light chain (MLC) to inhibit cofilin [78]. In addition, TGF-β affects
tight junctions through SMAD7-dependent CDC42-PAK1 (p21-activated kinase) and filopodia formation.
The TRAF6-TAK1-JNK/P38 pathway and PI3K-AKT-mTOR signaling are also necessary non-SMAD
pathways for TGF-β-mediated EMT [79,80]. Scientists have shown that PI3K/AKT signaling promotes
tumor metastasis by inducing TWIST1 phosphorylation, via a crosstalk between AKT/PKB and TGF-β
signaling [81]. Twist also had a significant effect on AKT signaling pathway activation by inducing
expression of miR-10b in gastric cancer cells, and the miR-10b induced by TWIST increased the expression
of a well-characterized pro-metastatic gene, RHOC [82]. Moreover, in an orthotopic syngeneic mouse tumor
model, metastasis caused by EMT was attenuated in mice treated with the p38 inhibitor SB203580 [83].
TRAF6 knockdown inhibited the migration and invasion caused by EMT of SCCHN (squamous cell
carcinoma of head and neck) cells [84].
In addition to these SMAD/non-SMAD pathways, TGF-β affects the activities of other EMT
trigger signaling pathways (NOTCH, WNT, INTEGRIN, etc.) by several complexes, such as ZEB1/2,
the SNAIL1-SMAD3/4 complex, the β-catenin-SMAD2 complex, the LEF1-SMAD3/4 complex and
the SMAD3-AP1-1 complex [85]. As early as 20 years ago, scientists discovered that SMAD4 could
form a complex with β-catenin and LEF1/TCF (lymphoid enhancer factor1), which are downstream
components of the Wnt signaling cascade in vivo [86]. SMAD signaling subsequently stimulates the
formation of β-catenin/LEF1 and SNAIL-LEF1 complexes, which promote EMT by inhibiting the
expression of E-cadherin [87,88]. TGF-β can promote EMT associated with WNT-11 signals through
the WNT-11 receptor FZD8 in prostate cancer [89]. Moreover, WNT-11 signaling mediates the nuclear
entry process of TAK1 (TGF-β-activated kinase) [90]. The TGF-β and NOTCH pathways coregulate
a large cohort of genes in human cancer, such as renal cell carcinoma [91]. R-SMAD activates the
NOTCH ligand Jagged1 to release Notch intracellular domain (ICN) and then binds to CLS (an acronym
for CBF-1/RBPJ-κ in Homo sapiens/Mus musculus respectively, Suppressor of Hairless in Drosophila
melanogaster, Lag-1 in Caenorhabditis elegans). This ICN-CLS complex induces the binding of the
transcription factor SNAIL or HEYl to the E-cadherin E-box to reduce E-cadherin expression and
initiate the EMT process [92]. Moreover, SMAD signaling and MAPK/JNK signaling converge at
AP1-binding promoter sites by SMAD3 and SMAD4, which cooperate with c-JUN/c-FOS [93], and the
RAS-ERK MAP kinase pathways are likely to act synergistically with TGF-β and contribute to multiple
aspects of the EMT, including the pro-invasive and pro-metastatic behavior of tumor cells of diverse
tissue origins [94]. TGF-β increases the level of SNAIL and promotes EMT with the cooperation of
oncogenic RAS [57] and the transcription factor nuclear factor κB (NF-κB) [95]. In addition, TGF-β
upregulates receptors and ligands of PDGF, leading to phosphorylation of PI3K and activation of the
SRC/STAT3 pathway, thereby triggering the EMT process [96].
5.3. MicroRNAs Involved in TGF-β-induced EMT

Two microRNA (a class of small noncoding RNAs approximately 22 nt in length)-dependent
negative feedback loops are at the heart TGF-β-induced EMT (Figure 4). These pathways are the
SNAIL1/miR-34 family/ZEB/miR-200 family feedback loop and the autocrine TGF-β/miR-200 feedback
loop [97].
316 5.3. MicroRNAs Involved in TGF-β-induced EMT
317 Two microRNA (a class of small noncoding RNAs approximately 22 nt in length)-dependent
318 negative feedback loops are at the heart TGF-β-induced EMT (Figure 4). These pathways are the
319Int. J. SNAIL1/miR-34 family/ZEB/miR-200 family feedback loop and the autocrine TGF-β/miR-2009 of 34
Mol. Sci. 2019, 20, 2767
320 feedback loop [97].
321
322 Figure 4. MicroRNAs in TGF-β-induced EMT. At the heart of TGF-β-induced EMT, there are two
Figure 4. MicroRNAs in TGF-β-induced EMT. At the heart of TGF-β-induced EMT, there are two
323 mainmain
double-negative
double-negative feedback
feedback regulatory
regulatoryloops
loops of
of miRNAs, e.g.,the
miRNAs, e.g., theSNAIL1/miR-34
SNAIL1/miR-34 family
family and and
324 ZEB/miR-200 family and the autocrine TGF-β/miR-200 negative feedback loop.
ZEB/miR-200 family and the autocrine TGF-β/miR-200 negative feedback loop. Specifically, TGF-β Specifically, TGF-β
325 downregulates miR-200 family members, thereby increasing ZEB1 and ZEB2 mRNA
downregulates miR-200 family members, thereby increasing ZEB1 and ZEB2 mRNA levels indirectly, levels indirectly,
326 and ZEB binds
and ZEB to promoters
binds to promoters ofofthethemiR-200
miR-200 members
members to torepress
represstheir
their expression,
expression, thus
thus constituting
constituting a a
327 double-negative
double-negative regulatory
regulatory loop.
loop. TheThesame
samesituation
situation occurs
occursininSNAIL1
SNAIL1 and
andmiR-34,
miR-34,which are directly
which are directly
328 linked
linked to status.
to p53 p53 status.
For For
the the autocrine
autocrine TGF-β/miR-200
TGF-β/miR-200 system,autocrine
system, autocrineTGF-β
TGF-β positively
positively regulates
regulates the
329 the expression
expression of SNAIL1of SNAIL1
and then and then increases
increases ZEB mRNA
ZEB mRNA and protein
and protein levels,levels, further
further downregulating
downregulating miR-200.
330 miR-200.
Inhibitory Inhibitory
signals signals are
are indicated with indicated with
inhibitory inhibitory
(dashed) red(dashed)
arrows; red arrows; Stimulatory
Stimulatory signals are signals
indicatedarewith
331 indicated with green arrows.
green arrows.
332 Mechanistically, TGF-β downregulates miR-200 family members, including miR-200a/-200b/-

Mechanistically, TGF-β downregulates miR-200 family members, including
333 200c/-141/-429, which augments ZEB1 and ZEB2 mRNA levels. ZEB counteracts this mechanism
miR-200a/-200b/-200c/-141/-429, which augments ZEB1 and ZEB2 mRNA levels. ZEB counteracts
334 through binding to the promoters of the miR-200 members and thereby repressing their expression.
335this mechanism through binding to the promoters of the miR-200 members and thereby repressing
Additionally, miR-200 family members maintain the epithelial phenotype not only by targeting
336theirZEB1/2
expression.
but alsoAdditionally, miR-200
by actively repressing family
genes members
involved maintain
in cell motility andthe epithelial
invasion phenotype not
[98]. MiR-1199-5p
337only by targeting ZEB1/2 but also by actively repressing genes involved
similarly regulates ZEB1 expression [99]. A comparable mechanism governs SNAIL1/miR-34 in cell motility
and the and
338invasion [98]. MiR-1199-5p similarly regulates ZEB1 expression [99]. A comparable
control of p53 status [100]. One study showed that in colorectal cancer, Zinc Finger protein 281 mechanism
339governs SNAIL1/miR-34
(ZNF281) and the control
can be an intermediate of p53
regulator status SNAIL1
between [100]. Oneandstudy
miR-34showed that
[101]. In in colorectal
addition to SNAILcancer,
340Zinc and p53,protein
Finger miR-34b281experiences
(ZNF281)epigenetic
can be anregulation (chromatin
intermediate modifications
regulator and DNAand
between SNAIL1 methylation)
miR-34 [101].
341In addition
by directly targeting
to SNAIL andmethyltransferases and deacetylases,
p53, miR-34b experiences epigeneticresulting in a positive
regulation (chromatin feedback loop
modifications
342and inducing partial demethylation and activity [102]. Silencing miR-34a promoted
DNA methylation) by directly targeting methyltransferases and deacetylases, resulting liver metastases of in a
343positive
colon cancer associated with upregulation of c-MET, SNAIL, and β-catenin expression [103].
feedback loop inducing partial demethylation and activity [102]. Silencing miR-34a promoted
344 Transcriptome profiling studies have demonstrated that TGF-β signaling regulates the SMAD4/miR-
liver metastases of colon cancer associated with upregulation of c-MET, SNAIL, and β-catenin
expression [103]. Transcriptome profiling studies have demonstrated that TGF-β signaling regulates
the SMAD4/miR-34a signaling network [104]. The SNAIL1/miR-34 regulatory loop was shown
to be involved in the early reversible stage of EMT (from epithelial to P-EMT), whereas the
ZEB/miR-200 system is responsible for the establishment of a mesenchymal state [105]. For the
autocrine TGF-β/miR-200 system, autocrine TGF-β positively regulates the expression of SNAIL1 and
then increases ZEB mRNA and protein levels, further affecting miR-200 [106]. This process makes the
second switch (from P-EMT to mesenchymal) irreversible, modulating the maintenance of EMT.
Int. J. Mol. Sci. 2019, 20, 2767 10 of 34
High mobility group protein A2 (HMGA2) has been shown to promote lung cancer progression
in mouse and human cells by competing with TGF-β type III receptor for the let-7 microRNA (miRNA)
family [107], while decreased let-7g levels influence the TGF-β pathway by targeting TβR1 and
SMAD2 gene expression [108]. The overexpression of miR-10b induced TGF-β-driven EMT in breast
cancer [109]. In contrast, silencing of miR-10b markedly suppressed the formation of lung metastases
by inhibiting its target gene HOXD10 in a mouse mammary tumor model [110].
TGF-β upregulates certain miRNAs, such as miR-182, which prolong NF-κB activation by
directly suppressing an NF-κB negative regulator (cylindromatosis, CYLD) [111]. Overexpression of
miR-182 restrained SMAD7 expression and promoted breast tumor invasion and TGF-β-induced
osteoclastogenesis and bone metastasis [73]. MiR-181a, another miRNA upregulated by TGF-β, promoted
TGF-β-mediated EMT and metastasis in breast cancer [112] and via repression of SMAD7 in ovarian cancer
progression [113]. TGF-β activates miR-1269 by SOX4 and thereby enhances TGF-β signaling by targeting
SMAD7 and HOXD10. This positive feedback loop significantly increased the ability of colorectal cancer
cells to invade and metastasize in vivo [114]. Overexpression of miR-216a/217 activated the PI3K/AKT and
TGF-β pathways by targeting PTEN, and SMAD7 underlies hepatocarcinogenesis and tumor recurrence
of hepatocarcinoma [115]. TGF-β induces the expression and promoter activity of miR-155 through
SMAD4. This change reduces RHOA protein and disrupts tight junction formation, leading to EMT [116].
Other research has shown that miR-206 inhibits autocrine production of TGF-β as well as downstream
neuropilin-1 (NRP1) and SMAD2 expression, resulting in decreased migration, invasion, and EMT in
breast cancer cells [117]. Several other miRNAs, such as miR-373, miR-655, miR206, miR-155, miR-140-5p,
miR-494, miR-125a/b, and miR-375, have been implicated in EMT [118–122]. However, for many of these
factors, their specific functions remain to be elucidated.
5.4. LncRNAs Involved in TGF-β-induced EMT

Long non coding RNAs (LncRNAs) are a class of RNAs that do not encode proteins or have minimal
coding capacity. LncRNAs are emerging as important regulators of a variety of cellular and physiologic
functions, such as chromatin dynamics, gene expression, growth, differentiation, and development [123].
LncRNAs can be differentially expressed and localized within the cell. They play a role in chromatin
and DNA interactions, negatively or positively affecting the stability or processing of coding mRNA,
directly binding to and modulating the functions of signaling proteins, and competitively binding to
and thereby controlling the function of miRNAs [124]. Aberrant expression and mutations in lncRNAs
have been linked to tumorigenesis, metastasis, and tumor stage [125]. Moreover, they have been
detected in the circulating blood and/or urine of cancer patients. For example, plasma levels of a novel
lncRNA, p53-induced transcript (Linc-pint), were significantly lower in patients with pancreatic ductal
adenocarcinoma (PDAC) than healthy controls [126]. The expression of twenty lncRNAs linked with
breast cancer-associated genes (BCAGs) was detectable in human breast cancer cell lines with different
expression patterns [127]. LncRNAs are novel, potential therapeutic targets and biomarkers for cancer
treatment, and new functions continue to be discovered [128].
Whereas previous studies have shown the involvement of miRNAs in regulating TGF-β signaling
and EMT, only a few studies have reported a prominent role of lncRNAs in these processes. Trans-acting
lncRNA ELIT-1 induced by TGF-β1 forms a positive TGF-β/SMAD3 signaling feedback and promotes
EMT progression by acting as a SMAD3 cofactor [129]. The miR-17~92 polycistronic miRNA cluster
encoded by the lncRNA MIR17HG locus was shown to attenuate the TGF-β signaling pathway and
stimulate angiogenesis and tumor growth [65,130]. In a recent study, researchers found that a lncRNA
activated by TGF-β, lncRNA-ATB, induces EMT and invasion by competitively binding miR-200 family
members, which promoted organ-specific metastasis by binding IL-11 mRNA. This competitive binding
increased IL-11 mRNA stability, which caused autocrine induction of IL-11 and subsequent activation
of STAT3 signaling [131]. These findings suggest that lncRNA-ATB, a mediator of TGF-β signaling,
could predispose HCC patients to metastasis [132]. Another study showed that lncRNA-PNUTS,
which is highly expressed in mesenchymal breast tumor cells, competitive binds to and neutralizes the
396 that a lncRNA activated by TGF-β, lncRNA-ATB, induces EMT and invasion by competitively
397Int. J. binding miR-200
Mol. Sci. 2019, family members, which promoted organ-specific metastasis by binding IL-11
20, 2767 11 of 34
398 mRNA. This competitive binding increased IL-11 mRNA stability, which caused autocrine induction
399 of IL-11 and subsequent activation of STAT3 signaling [131]. These findings suggest that lncRNA-
400activityATB,ofamiR-205
mediatorduring
of TGF-β EMT. Moreover,
signaling, couldelevated
predispose expression
HCC patients of lncRNA-PNUTS
to metastasis [132]. was correlated
Another
401withstudy showed that
upregulated lncRNA-PNUTS,
levels of ZEB mRNAs which is highly
[133]. LncRNA expressed
MEG3incan mesenchymal
modulate breast tumor of
the activity cells,
TGF-β
402genescompetitive binds to and neutralizes the activity of miR-205 during
by binding to distal regulatory elements [134]. Another lncRNA, DNM3OS, was associated withEMT. Moreover, elevated
403overexpression
expression of oflncRNA-PNUTS was correlated
TWIST1 and specifically with upregulated
contributed to EMT inlevels
ovarianof ZEB mRNAs
cancer [135].[133]. LncRNA
Recently, a paper
404showed MEG3 can modulate the activity of TGF-β genes by binding to distal
that lncRNA-MUF can directly activate WNT/β-catenin signaling and EMT by binding regulatory elements [134]. to
405 Another lncRNA, DNM3OS, was associated with overexpression of TWIST1 and specifically
ANNEXIN 2A. LncRNA-MUF can also indirectly promote EMT by competitively binding to miR-34a
406 contributed to EMT in ovarian cancer [135]. Recently, a paper showed that lncRNA-MUF can directly
and upregulating SNAIL1 expression [136].
407 activate WNT/β-catenin signaling and EMT by binding to ANNEXIN 2A. LncRNA-MUF can also
408 Reduced promote
indirectly lncRNAEMT H19 byexpression in hepatocarcinogenesis
competitively binding to miR-34a and (HCG) tissues from
upregulating SNAIL1patients with the
expression
409epithelial
[136]. TGF-β gene signature [137] but increased H19 expression promoted tumor metastasis after
410TGF-β treatment in Hep3B cells [138]. In a mouse model of spontaneous
Reduced lncRNA H19 expression in hepatocarcinogenesis (HCG) tissues from patients with the metastatic breast cancer,
411lncRNA H19 TGF-β
epithelial mediatedgeneEMT and [137]
signature METbut byincreased
differentially binding to
H19 expression the microRNAs
promoted miR-200b/c
tumor metastasis after and
412let-7bTGF-β[139].treatment
LncRNA in H19
Hep3Bcancells [138].
also In a mouse
interact modeland/or
with SLUG of spontaneous
EZH2, whichmetastatic breast E-cadherin
regulates cancer,
413expression
lncRNA[140].
H19 mediated
Lnc-Spry1 EMT is and MET by differentially
downregulated by TGF-β binding to the microRNAs
and plays miR-200b/c
a direct regulatory roleandin the
414earlylet-7b [139]. LncRNA H19 can also interact with SLUG and/or EZH2, which
stage of TGF-β-induced EMT, thus affecting cell invasion and migration. This molecule also regulates E-cadherin
415controlsexpression [140]. Lnc-Spry1 is downregulated by TGF-β and plays a direct regulatory role in the early
gene and protein expression levels through an interaction with the splicing factor U2AF65 [141].
416 stage of TGF-β-induced EMT, thus affecting cell invasion and migration. This molecule also controls
LncRNA-KRTAP5-AS1 and lncRNA-TUBB2A control the function of CLAUDIN-4 and thereby influence
417 gene and protein expression levels through an interaction with the splicing factor U2AF65 [141].
418EMTLncRNA-KRTAP5-AS1
in gastric cancer [142]. LncRNA HOTAIR (for HOX Transcript antisense intergenic RNA) acts as a
and lncRNA-TUBB2A control the function of CLAUDIN-4 and thereby
419crucial player EMT
influence during in EMT
gastricbycancer
mediating
[142]. aLncRNA
physicalHOTAIR
interaction
(for between SNAIL antisense
HOX Transcript and EZH2, which form
intergenic
420an enzymatic
RNA) actssubunit of the
as a crucial polycomb
player during repressive complex
EMT by mediating 2, the main
a physical writer of
interaction chromatin-repressive
between SNAIL and
421marks EZH2, which form an enzymatic subunit of the polycomb repressive complex 2, the mainsignaling
[143]. Together, these findings suggest that lncRNAs can be mediators of TGF-β writer of and
422maychromatin-repressive
serve as a potentialmarks target[143].
for anti-metastatic therapies.
Together, these findings The that
suggest mechanisms
lncRNAs can by be
which lncRNAs
mediators
423regulateof TGF-β
TGF-β signaling and are
signaling maylargely
serve asunknown,
a potential and
targethow
for anti-metastatic
they affect TGF-β therapies. The mechanisms
pathway components in
424cancer by metastasis
which lncRNAsremains regulate TGF-β signaling
to be discovered are 5largely
(Figure and Tableunknown,
1). and how they affect TGF-β
425 pathway components in cancer metastasis remains to be discovered (Figure 5 and Table 1).
426
Figure 5. LncRNAs in TGF-β signaling and TGF-β-induced EMT. LncRNAs associated with TGF-β
signaling and TGF-β-induced EMT in various cancer cell types. LncRNAs with high expression in
tumor tissues (in red) and low expression in tumor tissues (in green).
Int. J. Mol. Sci. 2019, 20, 2767 12 of 34
Table 1. LncRNAs involved in TGF-β signaling, focusing on those that impact TGF-β-induced EMT.
LncRNAs with high expression in tumor tissues (in red) and low expression in tumor tissues (in green).
Cancer Type LncRNA Function and Mechanism of Action Example of Key Findings or Experiments
Knockdown results in a morphological change of
Functions as a sponge of miR-141-3p, breast cancer cells from spindle-like to round shape
lncRNA-ATB [144]
increasing ZEB1 and ZEB2 expression. and in a remarkable inhibition of cell migration
and invasion.
Functions as an enhancer RNA (eRNA) to
activate Slug gene transcription in the Knockdown of either AC026904.1 or
nucleus, whereas UCA1 exerts a UCA1 prolongs survival time of the nude mice
AC026904.1 and UCA1 [145] competitive endogenous RNA (ceRNA) for bearing D3H2LN mammary tumors, these two
titrating miR-1 and miR-203a to promote genes exert critical roles in TGF-β-induced EMT
Slug expression at the post-transcriptional and promote invasion in metastatic breast cancer.
level in the cytoplasm.
Suppresses TGF-β-induced EMT by Overexpression reduces TGF-β-induced tumor
NKILA [146]
blocking NF-κB signaling metastasis in vivo.
Functions as a downstream effector Ectopic expression partly attenuates the
molecule, down-regulated by TGF-β1, TGF-β1-induced EMT and knockdown promotes
ANCR [147]
and is essential for TGF-β1-induced EMT TGF-β1-induced EMT and metastasis in breast
by decreasing RUNX2 expression. cancer.
Functions as an immediate-early regulator
of EMT that is downregulated by TGF-β,
Knockdown promotes a mesenchymal-like
affecting the expression of TGF-β-regulated
Lnc-Spry1 [141] phenotype and results in increased cell migration
gene targets; alternative splicing by
and invasion.
U2AF65 splicing factor; isoform switching
Breast cancer of fibroblast growth factor receptors.
Ectopic expression disrupts tight junction Knockdown results in decrease of cell migration,
lncRNA-HIT [148]
by targeting E-cadherin. invasion, tumor growth, and metastasis.
Functions as a sponge of miR-145 and
therefore upregulate the expression of Regulates the cancer stem cell phenotype in
linc-ROR [149,150] ARF6, which regulates adhesion and Triple-negative Breast Cancer, which plays a critical
invasion properties of breast tumor cells role in drug resistance and metastasis.
through E-cadherin.
Functions as an endogenous sponge of
miR-520c-3p, and controls the expression of Knockdown inhibits the progression of breast
HOXA-AS2 [151]
miR-520c-3p target genes, TβR2 and RELA, cancer cells in vitro and in vivo.
in breast cancer cells.
Knockdown causes cell cycle arrested in
G0/G1 phase, promotes cell apoptosis and
Down-regulation inhibits the proliferation,
CCAT2 [152] downregulates the protein expression levels
invasion and migration in breast cancer cells.
of TGF-β, Smad2 and α-SMA in breast
cancer cells.
Regulates the TGF-β pathway genes
through formation of RNA-DNA triplex Ectopic expression inhibits in vivo tumorigenesis
MEG3 [134,153,154] structures, and downregulates AKT and and angiogenesis in a nude mouse xenograft
functions as a sponge of miR-421 to model.
regulate E-cadherin expression.
LINC00978 (known as Knockdown inhibits the activation of Knockdown inhibits the proliferation, migration
MIR4435-2HG and TGF-β/SMAD signaling pathway and EMT and invasion and decreases the in vivo
AK001796) [155] in GC cells. tumorigenicity of GC cells in mice.
Knockdown inhibits TGF-β1-induced-EMT A potential oncogenic factor by regulating GC cells
UCA1 [156] process and the effect could be partly proliferation, invasion, and metastasis under
restored by TGF-β1 treatment. TGF-β1 induction.
Gastric cancer
A novel biomarker of lncRNA, correlated with
Induced by TGF-β and functions as a
lncRNA-ATB [157–159] increased invasion depth, more distant metastasis
ceRNA of miR-141-3p or miR-200s.
and advanced tumor-node-metastasis stage.
Functions as a competing endogenous
XIST [160] lncRNA (ceRNA) to regulate TGF-β1 by sh-XIST inhibited GC development in vitro.
sponging miR-185 in GC.
Int. J. Mol. Sci. 2019, 20, 2767 13 of 34
Table 1. Cont.
Regulates EMT process and the expression Overexpression inhibits cell viability, migration
LncRNA-LET [161] of TIMP2 and activates the Wnt/β-catenin and EMT process, and increases apoptosis in KGN
and Notch signaling pathways. cells
Functions by competing with miR-370-3p to Knockdown suppresses TGF-β-induced EMT,
H19 [162] regulate TGF-β-induced EMT in ovarian while H19 overexpression promotes
cancer. TGF-β-induced EMT.
Interrupts the interaction between
miR-200c/MALAT1 decreases the invasive capacity
TGF-β increases its expression by inhibiting
of EEC cells and EMT in vitro and inhibits EEC
miR-200c; MALAT1 interacts with
growth and EMT-associated protein expression
MALAT1 [163,164] MARCH7, which regulates
in vivo; This LncRNA plays an important role in
TGF-β-smad2/3 pathway by interacting
TβR2-Smad2/3-MALAT1/MARCH7/ATG7 feedback
with TβR2, via miR-200a as a ceRNA.
loop mediated autophagy, migration and invasion
Ovarian in ovarian cancer.
carcinomas Overexpression increases expression of TGF-β1 in
TGF-β1 treatment has no effects on LINK-A ovarian carcinoma cells and promotes cell
LINK-A [165] cervical cancer expression, and there is no clear migration and invasion and this effect can be
mechanism. attenuated by TGF-β1; Plasma levels are correlated
with distant tumor metastasis but not tumor size.
A promising prognostic marker that correlates with
lncRNA-ATB [166] No clear mechanism. the malignant phenotypes and poor prognosis of
cervical cancer
A mediator of TGF-β signaling.
Functions by competing with miR-25 and
Upregulation induces elevated SNAI2, which in
affecting SNAI2 to regulate the expression
PTAF [167] turn promoted OvCa cell EMT and invasion;
of many EMT-related protein-coding genes
knockdown inhibits tumor progression and
in OvCa.
metastasis in an orthotopic mouse model of OvCa.
Knockdown reverses CAF-CM-induced EMT and
Induced by TGF-β, and can regulate EMT
lncRNA-ZEB2NAT [168] invasion of cancer cells, as well as reduces the
process by affecting ZEB2 protein level.
ZEB2 protein level.
Overexpression is significantly correlated with
Induced by TGF-β and regulates EMT by poor survival in patients with bladder cancer.
negatively correlated E-cadherin, Inhibition of malat1 or suz12 suppresses the
MALAT1 [169]
N-cadherin and fibronectin expression by migratory and invasive properties induced by
zeste 12 (suz12) in vitro and in vivo. TGF-β and inhibits tumor metastasis in animal
models.
Its BMP-9-induced expression associates with
increased proliferation and migration of bladder
Induced by BMP9 through phosphorylated cancer cells. The promoting effect of BMP9 is
UCA1 [170]
Bladder cancer AKT and there are no clear mechanism. rescued after interfering with UCA1 in
BMP9 overexpressed bladder cancer cells both
in vitro and in vivo.
Is upregulated by TGF-β, acting as a
Its overexpression significantly promotes cell
lncRNA-ATB [171] molecular sponge of miR-126 and regulate
viability, migration, and invasion in T24 cells.
the direct target of miR-126 (KRAS).
Regulates the cell cycle, cyclin-D1 and EMT
PlncRNA-1 [172] in prostate cancer cells through the Functions as an oncogene.
TGF-β1 pathway.
Knockdown can reverse
High expression is associated with poor prognosis
TGF-β1-induced-EMT phenotype in
ROR [173] in gallbladder cancer patients and knockdown
SGC-996 and Noz cells. However, there are
inhibits cell proliferation, migration, and invasion.
no clear mechanism.
Regulates let-7a/TGF-β1/Smad signaling Overexpression promotes the proliferation and
ANRIL [174]
pathway. migration of prostate cancer cells.
Prostate cancer Upregulated by TGF-β, stimulates EMT Overexpression promotes, and knockdown of
associated with ZEB1 and lncRNA-ATB inhibits the growth of prostate cancer
lncRNA-ATB [175]
ZNF217 expression levels via ERK and cells via regulations of cell cycle regulatory protein
PI3K/AKT signaling pathways. expression levels.
Promotes the progression of pituitary tumors,
Activates the TGFBR2/SMAD3 and
ectopic expression of lnc-SNHG1 promotes cell
RAB11A/Wnt/β-catenin pathways in
lnc-SNHG1 [176] proliferation, migration, and invasion, as well as
pituitary tumor cells via sponging
the EMT, by affecting the cell cycle and cell
miR-302/372/373/520.
apoptosis in vitro and tumor growth in vivo.
Brain tumor
Functions as a ceRNA of miR-1 and
miR-203a to promote Slug expression, Knockdown attenuates EMT and stemness
UCA1 [177]
which underlies TGF-β-induced EMT and processes and their enhancement by TGF-β.
stemness of glioma cells.
Int. J. Mol. Sci. 2019, 20, 2767 14 of 34
Table 1. Cont.
Inhibits TGF-β-induced EMT and thereby
TGF-β1 inhibits its transcription in a
lncRNA-LINP1 [178] controlling cancer cell migration, invasion,
SMAD4-dependent manner.
and stemless in lung cancer cells.
Induced by TGF-β and functions via
Promotes non-small cell lung cancer progression
TBILA [179] cis-regulating HGAL and activating
in vitro and in vivo.
S100A7/JAB1 signaling.
Overexpression inhibits proliferation and
Functions as an endogenous sponge by
TGF-β1-induced EMT in A549 and H1299 cells,
directly binding to miR-137, negatively
XIST [180,181] regulating proliferation and TGF-β1-induced EMT
regulating its expression and regulating
in NSCLC, which could be involved in NSCLC
Notch gene expression.
progression.
Affects the physical interaction of its
binding partner (importin β1) with Smad3, Stimulates TGF-β signaling and regulates
NORAD [182]
and then inhibits the nuclear accumulation TGF-β-induced EMT-like phenotype in A549 cells.
of Smad complexes in response to TGF-β.
Regulates EMT by down-regulating
Overexpression promotes proliferation, migration,
miR-494 in A549 cells, which in turn
lncRNA-ATB [183] and invasion of A549 cells. In contract, ATB silence
increases phosphorylated levels of AKT,
shows the opposite influence.
JAK1, and STAT3.
Functions as a sponge of miR-150-5p and
Inhibition attenuates the tumorigenesis ability of
linc00673 [184] indirectly modulates the expression of key
A549 cells in vivo.
EMT regulator ZEB1.
Expression is regulated by TGF-β and Inhibits migration, invasion and viability of
Lung cancer regulates EMT process by inhibiting the NSCLC cells. Lower NKILA expression are
NKILA [185]
phosphorylation of IκBα and NF-κB correlated with lymph node metastasis and
activation to attenuate Snail expression. advanced TNM stage.
Knockdown inhibits TGF-β-mediated changes in
Associates with JARID2 and the regulatory
cell morphology and cell motility characteristic of
regions of target genes to recruit the
MEG3 [186] EMT and counteracts TGF-β-dependent changes in
complex by epigenetic regulation
the expression of EMT-related genes; In contrast,
(PRC2/JARID2/ H3K27 axis).
overexpression enhances these effects.
ls highly enriched in TGF-β-mediated
exosomes and might function by increasing Knockdown affects lung cancer invasion and
lnc-MMP2-2 [187]
the expression of MMP2 through its vascular permeability.
enhancer activity.
Inhibits NSCLC cell migration and invasion Low expression level indicates shorter
by downregulating TGF-β1 expression, postoperative survival time of NSCLC patients,
however TGF-β1 treatment shows no whereas, ectopic expression inhibits NSCLC cell
ANCR [188]
significant effects on ANCR expression but migration, invasion and downregulated
promotes NSCLC cell migration and TGF-β1 expression, and this effect can be
invasion. attenuated by TGF- β1.
Functions as a mediator of TGF-β signaling,
Knockdown promotes cell migration and invasion,
LINC01186 [189] is down-regulated by TGF-β1 regulating
whereas, overexpression prevents cell metastasis.
EMT by Smad3
Regulates the expression of various
Its expression may determine the overall survival
pathways and genes, especially
HOXA11-AS [190] and disease-free survival of lung adenocarcinoma
DOCK8 and TGF-β pathway, however,
patients in TCGA.
there is no clear mechanism.
Knockdown inhibits the proliferation, migration
Functions as a ceRNA of miR-140-5p and
lncRNA SBF2-AS1 [191] and invasion of HCC cells and attenuate the
regulates the expression of TβR1.
development of HCC tumor in vivo.
Ectopic expression in HCC cells promotes cell
Functions as a ceRNA of miR-26a/b and
proliferation and induces drug resistance, whereas
thereby modulates the expression of
SNHG6-003 [166] knockdown promotes apoptosis. High expression
transforming growth factor-β-activated
of SNHG6-003 closely correlated with tumor
kinase 1 (TAK1).
progression and shorter survival in HCC patients.
Liver cancer
Interacts with KRT19, as a sponge of Knockdown inhibits cell proliferation and invasion
LINC00974 [192] miR-642, activating the Notch and TGF-β with an activation of apoptosis and cell cycle arrest
pathways. both in vitro and in vitro.
Upregulated by TGF-b and can induce EMT
and invasion by acting as ceRNA of
miR-200 family and increasing ZEB1/2; Promotes the invasion-metastasis cascade in
lncRNA-ATB [132]
promotes organ colonization by binding hepatocellular carcinoma.
IL-11 mRNA, autocrine induction of IL-11,
and triggering STAT3 signaling.
Int. J. Mol. Sci. 2019, 20, 2767 15 of 34
Table 1. Cont.
Acts as an oncogene in pancreatic cancer,
Regulates EMT process via TGF-β1/Smad
PVT1 [193] knockdown of PVT1 inhibits viability, adhesion,
signaling.
migration and invasion.
MEG8, which shares the DLK1-DIO3 locus
with MEG3, can induce the recruitment of
EZH2 protein to miR-34a and
miR-203 genes for histone H3 methylation
Plays critical role in TGF-β-induced EMT in
MEG8 [194] and transcriptional repression, inducing
A549 lung cancer and Panc1 pancreatic cancer cells.
EMT-related cell morphological changes
and increases cell motility in the absence of
TGF-β by activating the gene expression
Pancreatic program required for EMT.
cancer Overexpression increases cell proliferation and
Regulates EMT process though
TUG1 [195] migration capacities, enhancing the proliferation
TGF-β/Smad pathway
and migration of pancreatic cancer cells
Induced by TGF-β, and this gene contains
miR-100, miR-125b and let-7a in its intron,
through SMAD2/3. These miRNAs regulate Plays prominent role in metastasis of pancreatic
MIR100HG [196]
a multitude of genes involved in the cancer.
inhibition of p53 and DNA damage
response pathways.
Low expression levels are correlated with lymph
lncRNA-ATB [197] No clear mechanism. node metastases neural invasion, and clinical stage
and worse overall survival prognoses of patients.
Upregulation reduces migration, invasion,
BX357664 [197,198] Blocks the TGF-β1/p38/HSP27 pathway.
and proliferation capabilities in RCC cells.
Its expression is correlated with metastases and
Renal cancer promotes cell migration and invasion in renal cell
carcinoma. Knockdown could inhibit cell
lncRNA-ATB [199] No clear mechanism.
proliferation, trigger apoptosis, reduce
epithelial-to-mesenchymal transition program and
suppress cell migration and invasion.
High expression is significantly associated with
Upregulated by TGF-β, and suppresses
greater tumor size, depth of tumor invasion,
lncRNA-ATB [199–201] E-cadherin expression and promoting EMT
lymphatic invasion, vascular invasion, and lymph
process.
node metastasis.
Colorectal Downregulated by TGF-β and its
cancer expression is positively correlated with
Inhibits EMT and metastasis in colorectal cancer
E-cadherin, and negatively correlated with
LINC01133 [202] (CRC) cells and low LIINC01133 expression in
Vimentin. Directly interacting with SRSF6,
tumors with poor survival in CRC samples.
which promotes EMT and metastasis in
CRC cells.
Represses Notch and TGF-β signaling
pathway by inhibiting Notch1, Hes1, Overexpression represses cell proliferation and
MEG3 [203]
TGF-β and N-cadherin expression, migration ability.
and increasing E-cadherin.
Induced by TGF-β and its overexpression
Overexpression promotes cell metastasis,
decreases E-cadherin level, however, this
Bone tumor MALAT1 [204] a potential diagnostic and prognostic factor in
effect was partially reversed by
osteosarcoma.
EZH2 knockdown.
Inhibits tumor suppressor miR-34s signals
and the targets of miR-34a, including
Knockdown delays the tumor growth in
SNHG7 [205] proliferation-related Notch1,
osteosarcoma tissues.
apoptosis-related BCL-2, cell cycle-related
CDK6, and EMT-related SMAD4.
Induced by TGF-β and supports a role for Functions as an oncogene and as a tumor
MALAT1 [206]
MALAT1 in EMT in thyroid tumors. suppressor in different types of thyroid tumors.
Functions as a competing endogenous RNA Expression is induced by TGF-β in cells
ROR [207]
(ceRNA) of sponging miR-145. undergoing EMT.
A novel prognostic factor, which correlates with
Knockdown increases the levels of
poor prognosis and exhibits that silenced
Thyroid cancer SPRY4-IT1 [208] TGF-β1 and p-Smad2/3 and this effect could
SPRY4-IT1 inhibited the proliferative and
be rescued by the interference of TGF-β1.
migratory abilities of TC cells.
Promotes invasion and metastasis of TC cells,
Reduces p15INK4B expression through
ANRIL [209] and the silencing of ANRIL inhibits the invasion
inhibiting TGF-β/Smad signaling pathway.
and metastasis of TPC-1 cells.
Int. J. Mol. Sci. 2019, 20, 2767 16 of 34
6. TGF-β and Metastasis
6.1. TGF-β-induced Metastasis in Tissues

Tumor metastasis is the primary cause of cancer lethality; it is a progressive, multifactorial and
multistep dynamic process, including detachment from a primary tumor, invasion into surrounding
tissues, invasion into blood circulation/lymphatic circulation, survival in the circulatory system,
extravasation from blood vessels, distal colonization, etc. [210]. The migration of cancer cells is pivotal
to early metastasis, and changes in tumor cells (acquired by EMT) and the microenvironment are
the two main factors that help cancer stem cells (CSCs) to escape from the primary site [211]. In the
tumor microenvironment, TGF-β, Chemokine 4/12 (CXCL4/12), interleukin-6 (IL-6) and tumor necrosis
factor-α (TNF-α), etc. can enhance EMT. At the same time, tumor cells secrete more epithelial growth
factors, fibroblast growth factor (FGF) and insulin-like growth factor (IGF), leading to a hypoxic,
acidic, high interstitial fluid pressure (IFP) state in the microenvironment, which activates cancer
associated fibroblasts (CAFs) to produce more matrix metalloproteinases (MMPs) and remodel the
tumor extracellular matrices (ECM) [212].
TGF-β induces metastasis to bone, liver, lung and other tissues of specific cancer types, such as
breast, lung, gastric and prostate cancers (Figure 6A) [213]. Metastatic cancer cells have been shown to
disturb the tight balance of bone transformation by osteoblasts and osteoclasts, conferring a receptive
outgrowth microenvironment [214]. Tumor cells secrete cytokines and parathyroid hormone-related
protein (PTHrP), which is the main inducer of osteoclast formation (also interleukin (IL)-1/-6/-11, etc.),
and its expression is specific to the bone metastasis microenvironment [215]. TGF-β released in the
active form upon osteoclastic bone resorption enhances PTHrP signaling in osteoblasts, resulting in
osteoblasts expressing receptor activator of NFκB ligand (RANKL) while reducing osteoprotegerin
(OPG) expression [216]. The high RANKL/OPG ratio enhances the osteolytic activity, which is
associated with the release of high levels of active TGF-β. This TGF-β can further upregulate PTHrP
expression by cancer cells, thereby forming a positive feedback loop called a “vicious cycle” [217].
Many specific studies have analyzed the role of TGF-β in lung and liver metastases. In a previous
study, increased TGF-β levels were shown to lead to lung metastases in the MMTV/PyVmT transgenic
model of metastatic breast cancer [218]. In addition, in breast cancer cells, TGF-β induced ANGPTL4 via
the SMAD signaling pathway, and this cytokine could disrupt lung capillary walls and seed pulmonary
metastases [219]. Because there are inherent differences in the microvasculature of these two tissues,
lung metastasis requires robust extravasation functions provided by ANGPTl4 and other factors and
additional lung colonizing functions achieved by ID1/ID3 [220]. Therefore, the vasculature disruptive
mechanism provides a selective invasive advantage in the lung but not bone. Another study showed
that the WNT signaling inhibitor Dickkopf 1 (DKK1) is a key factor for this metastatic preference; it
reduces the recruitment of macrophages and neutrophils by the WNT/PCP-RAC1-JNK pathway and
inhibits the level of tumor-derived TGF-β to inhibit lung metastasis. Thus, tumor cells that highly
secrete DKK1 tend to metastasize to bone, while those with low DKK1 secretion tend to metastasize to
the lung [221].
Statistical analysis of gene expression profiles revealed that TGF-β signaling is the most significant
gene pathway in liver metastases of colorectal cancer [222]. Exosomes (small membrane vesicles with
a size ranging from 40 to 100 nm) from pancreatic cancer induce Kupffer cells to release more TGF-β,
which in turn activates the fibrotic pathway and forms a proinflammatory environment that supports
pancreatic cancer metastasis [223].
Int. J. Mol. Sci. 2019, 20, 2767 17 of 34
459
460 Figure
Figure 6. TGF-β mediates
6. TGF-β mediates metastasis.
metastasis. TGF-βTGF-βproduced
producedbyby cancer
cancer cells
cells can
can alter
alter the
the bone
bone
461 microenvironment by inducing the expression of osteolytic factors like PTHrP and
microenvironment by inducing the expression of osteolytic factors like PTHrP and IL11. The IL11. The osteoclast
462 bone resorption
osteoclast bone via RANKLvia
resorption production
RANKL by osteoblasts
production byresults in more
osteoblasts release
results in of TGF-β,
more which
release in turn
of TGF-β,
463 acts on tumor cells thereby creating a positive feedback loop called a “vicious cycle”. Chemokine
which in turn acts on tumor cells thereby creating a positive feedback loop called a “vicious cycle”. receptor
464 CXCL4 and receptor
Chemokine angiogenesis
CXCL4 inducer connective tissue
and angiogenesis growth
inducer factor (CTGF)
connective are alsofactor
tissue growth key modulators
(CTGF) are
465 induced
also key modulators induced by TGF-β in this process. Another important factor of this is
by TGF-β in this process. Another important factor of this metastatic preference the Wnt
metastatic
antagonist Dickkopf 1 (DKK1); cells that highly secrete DKK1 tend to metastasize to bone, while low
466 preference is the Wnt antagonist Dickkopf 1 (DKK1); cells that highly secrete DKK1 tend to
DKK1-secreting tumor cells tend to metastasize to the lung. TGF-β leads to transcriptional upregulation
467 metastasize to bone, while low DKK1-secreting tumor cells tend to metastasize to the lung. TGF-β
of proangiogenic factors, including CTGF, matrix metalloprotease (MMP)2, MMP-9, and MMP10,
468 leads to transcriptional upregulation of proangiogenic factors, including CTGF, matrix
or inhibition of TIMP to mediate the formation of new blood vessels. TGF-β inhibits the proliferation
469 metalloprotease (MMP)2, MMP-9, and MMP10, or inhibition of TIMP to mediate the formation of new
of T cells and B cells and inhibits the production of immune factors by B lymphocytes. In addition,
470 blood vessels. TGF-β inhibits the proliferation of T cells and B cells and inhibits the production of
TGF-β-induced angiopoietin-like 4 (ANGPTL4) via the SMAD signaling pathway is proangiogenic and
471 immune factors by B lymphocytes. In addition, TGF-β-induced angiopoietin-like 4 (ANGPTL4) via
can disrupt lung capillary walls and seed pulmonary metastases. Inhibitory signals are indicated with
472 the SMAD signaling pathway is proangiogenic and can disrupt lung capillary walls and seed
inhibitory red arrows; Stimulatory signals are indicated with green arrows.
473 pulmonary metastases. Inhibitory signals are indicated with inhibitory red arrows; Stimulatory
474 signalsPromotes
6.2. TGF-β are indicated with green arrows.
Angiogenesis
475 Regardless
Many specific of the primary
studies haveor secondary
analyzed thetumor,
role of angiogenesis
TGF-β in lungoccurs once
and liver the tumor In
metastases. is amore than
previous
476 1–2 mm in diameter; therefore, a rich blood supply is necessary to
study, increased TGF-β levels were shown to lead to lung metastases in the MMTV/PyVmT provide nutrients and oxygen for
477 tumor growth
transgenic andofmetastasis
model metastatic[224].
breast Tumor
cancercells secrete
[218]. a varietyinofbreast
In addition, growth factors,
cancer including
cells, TGF-β,
TGF-β induced
478 to accelerate the development of cancer by inducing angiogenesis (Figure
ANGPTL4 via the SMAD signaling pathway, and this cytokine could disrupt lung capillary walls 6B) [225].
479 On endothelial
and seed pulmonarycells TGF-β can
metastases [219].bind to the there
Because type Iare
receptor, activin
inherent receptor-like
differences kinase 1 (ALK-1),
in the microvasculature
480 prompting downstream signaling involving intracellular and nuclear
of these two tissues, lung metastasis requires robust extravasation functions provided by proteins (SMADs andANGPTl4
Id1) and
481 leading to a proangiogenic response [226]. Furthermore, TGF-β1 and hypoxia
and other factors and additional lung colonizing functions achieved by ID1/ID3 [220]. Therefore, the are potent inducers
482 of vascular endothelial
vasculature growth factor
disruptive mechanism (VEGF)
provides expression
a selective in tumor
invasive cells, and
advantage oncogenes,
in the lung butespecially
not bone.
483 RAS,
Another study showed that the WNT signaling inhibitor Dickkopf 1 (DKK1) is a key for
can also combine with the tumor microenvironment, providing the foundation factortumor cell
for this
484 invasion and angiogenesis [227]. Using a mouse mammary carcinoma model,
metastatic preference; it reduces the recruitment of macrophages and neutrophils by the WNT/PCP- researchers confirmed
485 that VEGF expression
RAC1-JNK pathway andin peri-necrotic
inhibits theareas
levelisofsynergized
tumor-derivedby both hypoxia
TGF-β and TGF-β1,
to inhibit further showing
lung metastasis. Thus,
486 that
tumor cells that highly secrete DKK1 tend to metastasize to bone, while those with associating
this cooperation is achieved through hypoxia-inducible factor (HIF)-1α physically low DKK1
487 with SMAD3
secretion tend[228]. For example,
to metastasize to theTGF-β increases the expression of VEGF-C by coordinating with
lung [221].
488 sine oculis
Statistical analysis of gene expressiontumor
homeobox homolog 1 (SIX1) in cells,revealed
profiles promoting thattumor
TGF-β lymph angiogenesis
signaling is the mostand
489 significant gene pathway in liver metastases of colorectal cancer [222]. Exosomes (small membrane
490 vesicles with a size ranging from 40 to 100 nm) from pancreatic cancer induce Kupffer cells to release
Int. J. Mol. Sci. 2019, 20, 2767 18 of 34
lymph node metastasis [229]. Likewise, TGF-β1 could promote macrophages to secrete more VEGF
via the TβRII/SMAD3 signaling pathway in oral squamous cell carcinoma [230]. TGF-β and VEGF
form a feedback loop through the Semaphorin3A/NEM axis; in general, abrogated VEGF inhibits
the endothelial cell paracrine TGF-β1 and endothelial SMAD2/3 activation; in turn, TGF-β1 further
stimulates endothelial Semaphorin3A expression [231]. Studies have shown that in RAS-transformed
epithelial tumors, TGF-β significantly increases the expression of VEGF/VEGF-R, which has a powerful
effect on capillary formation and migration of endothelial cells, thereby promoting angiogenesis in
tumor cells [232]. TGF-β mediates the formation of new blood vessels by promoting connective tissue
growth factor (CTGF) and angiogenic regulatory enzymes, such as matrix metalloproteinases (MMP-2,
MMP-9, MMP-10, etc.) or by inhibiting tissue inhibitor of metalloproteinases (TIMP) [233].
6.3. TGF-β Promotes Immune Evasion

Under physiological conditions, the immune system is the most important element of human
defense against cancer, and T lymphocytes and natural killer cells can recognize and specifically clear
tumor cells [4,234]. However, tumor cells evade this immune surveillance through immune evasion
of TGF-β, but the cellular mechanism by which TGF-β induces T cell dysfunction remains unclear.
TGF-β may inhibit the proliferation of T cells and B cells and inhibit the production of immune factors
by B lymphocytes (Figure 6C). In transgenic mouse studies, CD4+ and CD8+ T lymphocytes showed
that expression of dominant-negative TβR2 was more effective in clearing thymoma and melanoma
cells than in wild-type mice, which indicates that T lymphocytes are central targets for the negative
regulation of TGF-β [235]. Interestingly, T cell production of TGF-β1 was shown to be a requirement
for tumors to evade immune surveillance independent of TGF-β produced by tumors [236].
For example, regulation of tumor metastasis by TGF-β/SMAD signaling was found to be achieved
by impairing the activity of tumor-infiltrating T cells [237,238], i.e., the infiltration level of CD3+,
CD4+ and CD8+ cells and the proliferation and activity of T cells (such as the secretion of granzyme,
FAS ligand (FASL), perforin and interferon (IFN)-γ) [239,240]. Regulatory T-cells (Tregs), whose
excessive function inhibit antitumor immune responses, are another vital factor for TGF-β-mediated
immune evasion by suppressing the proliferation and activation of CD8+ cytotoxic T-cells [241].
Effector Treg cells express high amounts of integrin αvβ8, which enables them to activate latent
TGF-β, and tumor-derived TGF-β, in turn, induces FoxP3 expression and generates induced Treg
cells [242]. Tregs can produce cell surface docking receptors for latent TGF-β, called glycoprotein A
repetitions predominant (GARP). Further experiments revealed that overexpression of GARP leads
to more TGF-β-releasing Treg cells and enhanced TGF-β signaling, tumor growth and metastasis in
immunodeficient mice [243].
Additionally, TGF-β blocks immune surveillance by inhibiting migration and inducing apoptosis
of antigen-presenting cells, such as dendritic cells (DCs), whose function is to mature and stimulate T
cells during the immune response. Studies have shown that tumor-derived TGF-β significantly inhibits
the proliferation of human CD4+ T cells activated by dendritic cells [244–246]. TGF-β also has an
impact on myeloid cell functions. TGF-β in the tumor microenvironment polarizes tumor-associated
macrophages (TAMs) from the pretumor (M2) phenotype to the antitumor (M1) phenotype. TGF-β
inhibits neutrophil activity (i.e., degranulation) [247]. Furthermore, tumor-derived TGF-β polarizes the
tumor-associated neutrophil (TAN) phenotype from N1 to N2 and induces a population of protumor
TANs [248].
Moreover, the development of natural killer (NK) cells and T helper 1 (Th1) differentiation depend
on TGF-β signaling. Functional studies have demonstrated that selective deletion of SMAD4 in
NK cells impedes NK cell homeostasis and maturation, thereby reducing murine cytomegalovirus
clearance [249]. A TGF-β-regulated transcription factor, T-bet, is responsible for Th1 differentiation and
survival of activated CD4+ T cells via mediating CD122 expression and IL-15 signaling in Th1 cells [250].
Int. J. Mol. Sci. 2019, 20, 2767 19 of 34
7. Targeting the TGF-β Signaling Pathway in Cancer

Several TGF-β signaling inhibitors have been developed to curtail the aberrant TGF-β signaling
characteristics of tumors. There are several types of TGF-β drugs in (pre)clinical development: ligand
traps, antisense oligonucleotides (AONs), neutralizing antibodies, receptor domain-immunoglobulin
fusions and receptor kinase inhibitors (Figure 7) [251]. While these agents have shown promise in the
clinic, the complexity and pleiotropic nature of TGF-β tumor regulation render TGF-β targeted therapy
a challenge. Related to this, careful administration/dosing of TGF-β therapies as well as judicious
patient selection is needed to overcome on-target and off-target toxic side-effects [252].
Antisense oligonucleotides have been incorporated into immune cells to block translation or to
degrade TGF-β mRNA. Belagenpumatucel-L (Lucanix) is a therapeutic vaccine for non-small cell lung
cancer (NSCLC), which inhibits the expression of TGF-β2, thereby reducing the immunosuppressive
effect of TGF-β2 and thus enhancing its antitumor effect [253]. Trabedersen (AP 12009) is an antisense
oligonucleotide that specifically targets human TGF-β2 mRNA and has been used in the treatment
of metastatic melanoma and pancreatic cancer [254]. Its clinical development has been put on hold
(Clinical Trials: NCT00761280).
Multiple TβRI kinase inhibitors have been developed for the treatment of cancer and are under
clinical trials. For example, a phase I study of Galunisertib (LY2157299), a TβR1 kinase inhibitor, showed
an acceptable tolerability and safety profile in Japanese patients with advanced solid tumors [255] and
is currently under clinical development in combination with immunotherapy, anti-PD-1 antibodies,
including nivolumab and durvalumab (Clinical Trials: NCT02423343). SM16 (a TβRI kinase inhibitor)
and 1D11 (a TGF-β neutralizing antibody) synergistically inhibited metastasis in combination with
the agonistic OX40 antibody [256]. SD208, an inhibitor of TβR1, blocks the TGF-β/SMAD pathway,
Matrigel invasion and expression of TGF-β target genes (PTHrP, IL-11, CTGF, and RUNX2, etc.) and
was effective at preventing the development of bone metastases and decreasing the progression of
established osteolytic lesions in melanoma and glioma models [257,258]. However, another study
showed that SD-208 could not significantly reduce tumor growth and angiogenesis in SW-48 cells,
a human colorectal cancer model, and thus, its efficiency still needs to be assessed [259].
Blockade of TGF-β ligand and receptor binding is a crucial mechanism for TGF-β inhibitory
targeting agents. In a clinical phase I trial, PF-03446962, an anti-ALK1 antibody that displays
(dose-dependent) antiangiogenic activity, was tested. This antibody was administered to patients
who were resistant to anti-VEGF/VEGFR therapy [260]. However, in phase II, trials were stopped due
to ineffectiveness (NCT01620970). Another TGF-β monoclonal antibody, Fresolimumab (GC-1008),
was demonstrated to be safe and well tolerated in Phase I and Phase II trials [261]. Fresolimumab is an
anti-TGF-β neutralizing antibody capable of neutralizing all human isoforms of TGF-β [262]. John et
al. have shown that some melanoma or renal cancer patients with multiple doses of fresolimumab
treatment experienced cutaneous lesions during phase 1 clinical trials [195]. Moreover, a phase I study
noted that the maximum tolerated dose for the anti-TβRII monoclonal antibody LY3022859 could not
be determined, but dose escalation beyond 25 mg was considered unsafe in patients with advanced
solid tumors [263].
Finally, an interesting paper showed that a TGF-β inhibitor in combination with a PD-L1 inhibitor
(Atezolizumab) may be able to remodel the matrix microenvironment and allow T cells to enter the
interior of the tumor [237]. Furthermore, for chimeric antigen receptor (CAR) T cell therapies, CAR-T
cells can reverse the immunosuppressive effect of TGF-β [264]. This finding suggests that combination
therapies rather than mono-TGF-β therapies could prove to be the most effective approach in the future.
596 Finally, an interesting paper showed that a TGF-β inhibitor in combination with a PD-L1
597 inhibitor (Atezolizumab) may be able to remodel the matrix microenvironment and allow T cells to
598 enter the interior of the tumor [237]. Furthermore, for chimeric antigen receptor (CAR) T cell
599 therapies, CAR-T cells can reverse the immunosuppressive effect of TGF-β [264]. This finding
600 suggests
Int. that
J. Mol. Sci. combination
2019, 20, 2767 therapies rather than mono-TGF-β therapies could prove to be the20most
of 34
601 effective approach in the future.
602
Figure 7. Targeting the TGF-β signaling pathway in cancer. Various molecular mechanisms by
603 Figure TGF-β
which 7. Targeting the TGF-β
signaling signaling
is targeted for pathway
therapeuticin cancer. Various
gain are molecular
depicted. TGF-βmechanisms by which
inhibitory targeting
604 TGF-β signaling is targeted for therapeutic gain are depicted. TGF-β inhibitory targeting agents
agents (Table 1), including TβRI kinase inhibitors, AON targeting ligand or receptor gene expression,(Table 1),
605 including TβRI kinase inhibitors, AON targeting ligand or receptor gene expression, and antibodies
and antibodies interfering with ligand-receptor interactions, have been developed to curtail excessive
606 interfering
TGF-β with activation.
pathway ligand-receptor interactions,
Inhibitory have
signals are been with
indicated developed to curtail
inhibitory excessive
red arrows; TGF-β
Stimulatory
607 pathway
signals areactivation.
indicated Inhibitory
with greensignals
arrows.are indicated with inhibitory red arrows; Stimulatory signals
608 are indicated with green arrows.
8. Concluding Remarks
609 8. Concluding Remarks
TGF-β acts as a tumor suppressor during the early phase of cancer progression and as a tumor
610 TGF-β
promotor in acts as a tumor
advanced suppressor
stages. The latter during
role, in the early phase
particular, of cancer
has been progression
targeted and therapeutic
as a potential as a tumor
611 promotor toininhibiting
approach advancedtumorstages. The and
growth latterdissemination.
role, in particular,
However,hasdeveloping
been targeted as atreatments
effective potential
612 therapeutic
is approach by
severely hampered to inhibiting tumor
the biphasic role growth
of TGF-β andin dissemination. However,
cancer and its function indeveloping effective
many physiological
613 treatments including
processes, is severelythehampered by the system.
cardiovascular biphasicThere
role ofhas
TGF-β
beenin cancer
some and itsinfunction
progress in many
the treatment of
614 physiological processes, including the cardiovascular system. There has been some progress
liver cancer and other types of cancer, but there is a pressing need for a greater understanding of in the
615 treatment ofTGF-β
pathological liver mechanisms
cancer and other
as welltypes of cancer,
as greater but there protocols
clarity regarding is a pressing needadministration
of therapy for a greater
616 understanding
and of pathological TGF-β mechanisms as well as greater clarity regarding protocols of
patient selection.
617 therapy
Withadministration
the success of and patient
immune selection.inhibitors for cancer therapy and considering the potent
checkpoint
immune suppressive effects of TGF-β, it may be of particular interest to see whether TGF-β targeting
agents can increase the efficiency and range of immune therapeutic agents. Such trials are underway,
and the results are eagerly awaited. As highlighted in this review, other approaches could include the
targeting of E3 ubiquitin ligases and deubiquitinating enzymes and miRNAs and lncRNAs, which
control the stability of TGF-β receptors and SMAD proteins.
Author Contributions: Writing—original draft preparation, Y.H.; writing—review and editing, D.B.; supervision,
P.t.D.
Funding: Our studies on TGF-β signaling in cancer are supported by Cancer Genomics Centre Netherlands
(CGC.nl). YH is supported by CSC scholarship.
Int. J. Mol. Sci. 2019, 20, 2767 21 of 34
Acknowledgments: Our studies on TGF-β signaling in cancer are supported by Cancer Genomics Centre
Netherlands (CGC.nl). YH is supported by CSC scholarship.
Conflicts of Interest: The authors declare no conflict of interest.
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© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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International Journal of

Int. J. Mol. Sci. 2019, 20, 2767; doi:10.3390/ijms20112767 www.mdpi.com/journal/ijms

2. TGF-β and Signaling Transduction across the Plasma Membrane

3. Intracellular SMAD and Non-SMAD Signaling Pathways

4. Tumor Suppressive Effects of TGF-β

5. TGF-β-induced Tumor Promoting Effects

5.1. Cell Biology of TGF-β-induced EMT

5.3. MicroRNAs Involved in TGF-β-induced EMT

332 Mechanistically, TGF-β downregulates miR-200 family members, including miR-200a/-200b/-

5.4. LncRNAs Involved in TGF-β-induced EMT

6. TGF-β and Metastasis

6.1. TGF-β-induced Metastasis in Tissues

6.3. TGF-β Promotes Immune Evasion

7. Targeting the TGF-β Signaling Pathway in Cancer

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