Leading Edge

Review

EMT: 2016
M. Angela Nieto,1,6,* Ruby Yun-Ju Huang,2,3,6 Rebecca A. Jackson,4,6 and Jean Paul Thiery3,4,5,6,*
1Instituto de Neurociencias CSIC-UMH, Avda. Ramon y Cajal s/n, 03550 San Juan de Alicante, Spain
2Department of Obstetrics & Gynaecology, National University Hospital, Singapore 119228, Singapore
3Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599, Singapore
4Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117596, Singapore
5Institute of Molecular and Cell Biology, A-STAR, Singapore 138673, Singapore
6Co-first authors

*Correspondence: anieto@umh.es (M.A.N.), bchtjp@nus.edu.sg (J.P.T.)

http://dx.doi.org/10.1016/j.cell.2016.06.028

The significant parallels between cell plasticity during embryonic development and carcinoma pro-
gression have helped us understand the importance of the epithelial-mesenchymal transition (EMT)
in human disease. Our expanding knowledge of EMT has led to a clarification of the EMT program
as a set of multiple and dynamic transitional states between the epithelial and mesenchymal phe-
notypes, as opposed to a process involving a single binary decision. EMT and its intermediate
states have recently been identified as crucial drivers of organ fibrosis and tumor progression,
although there is some need for caution when interpreting its contribution to metastatic coloniza-
tion. Here, we discuss the current state-of-the-art and latest findings regarding the concept of
cellular plasticity and heterogeneity in EMT. We raise some of the questions pending and identify
the challenges faced in this fast-moving field.

Introduction transformation to transition more than a decade ago (first

During embryonic development, cells can transition between
epithelial and mesenchymal states in a highly plastic and dy-
namic manner. A shift toward the mesenchymal state, in a pro-
cess referred to as epithelial-mesenchymal transition (EMT),
modifies the adhesion molecules expressed by the cell, allow-
ing it to adopt a migratory and invasive behavior. The reverse
of this processmesenchymal-epithelial transition (MET)is
associated with a loss of this migratory freedom, with cells
adopting an apico-basal polarization and expressing the junc-
tional complexes that are hallmarks of epithelial tissues (Thiery
et al., 2009).
The EMT is executed in response to pleiotropic signaling fac-
tors that induce the expression of specific transcription factors
(TFs) called EMT-TFs (e.g., Snail, Zeb, Twist, and others) and
miRNAs together with epigenetic and post-translational regula-
tors, many of which are involved in embryonic development,
wound healing, fibrosis, and cancer metastasis. Indeed, the sig-
nificant parallels between cell plasticity in embryonic develop-
ment and carcinoma progression led to the proposition that
EMT is a central driver of epithelial-derived tumor malignancies
(Cano et al., 2000; Thiery, 2002). EMT has since been shown to
trigger the dissociation of carcinoma cells from primary carci-
nomas, which subsequently migrate and disseminate to distant
sites. Significantly, it is the MET that is then believed to trigger
the cessation of migration, inducing the same cells to proliferate
and seed the new tumor.
The classic description of EMT as the transformation of epithe-
lial cells into mesenchymal cells (Hay, 1995) may have invoked
the perception of this process as a shift between two alternative
states, mesenchymal or epithelial. However, the change from
meeting of The Epithelial-Mesenchymal Transition International
Association [TEMTIA]) already conveyed the concept of plas-
ticity and transitional states. It is therefore not surprising that
more recent work points to a greater flexibility in this transitional
process, and cells are no longer thought to oscillate between the
full epithelial and full mesenchymal states, but rather, they move
through a spectrum of intermediary phases. This purported plas-
ticity means that cells could linger in intermediary stages and
that they may frequently undergo a partial EMT program
(Figure 1). Much of the work into the mechanisms regulating
EMT has been carried out on cultured cells. However, the iden-
tification of hybrid intermediate EMT states during organ fibrosis
and in circulating tumor cells provides evidence that the spec-
trum of intermediary phases previously observed in cultured
cells reflects what happens in vivo.
In this review, we will discuss these intermediate states of
EMT, assessing whether the acquisition of stemness depends
on EMT and exploring some of the latest findings regarding
EMT in development and disease. We will also deliberate on
the impetus to exploit EMT for therapeutic gain and pose some
pending questions and challenges in this expanding field.
Broadening the Reductionists View: Intermediate EMT
Phenotypes
From a reductionists viewpoint, the operational definition of EMT
describes the loss and gain of certain characteristics during a sin-
gle transition between two states. As such, most experimental
models tend to consider a dramatic change in the expression
of selected epithelial and mesenchymal markers to confirm
EMT (Thiery and Sleeman, 2006): E-cadherin, occludins, and

Cell 166, June 30, 2016 2016 Elsevier Inc. 21


cytokeratins are the most commonly used markers for the epithe-
lial trait and N-cadherin and vimentin for the mesenchymal (Thiery
et al., 2009). This oversimplification ultimately led to some confu-
sion and considerable debate as to whether EMT actually occurs
in some circumstances, such as during carcinoma progression
(Tarin et al., 2005). However, the definition of EMT has now
been broadened, based on many observations and particularly
those regarding the so-called partial EMT. A partial EMT state
has been noted in association with many developmental, wound
healing, fibrosis, and cancer processes (Arnoux et al., 2008;
Blanco et al., 2007; Futterman et al., 2011; Grande et al., 2015;
Leroy and Mostov, 2007; Lovisa et al., 2015; Grigore et al.,
2016), evident through the existence of intermediate hybrid
epithelial and mesenchymal phenotypes (Abell et al., 2011;
Huang et al., 2013; Jordan et al., 2011; Yu et al., 2013).
Cells bearing this hybrid phenotype have been referred to as
metastable (Lee et al., 2006; Tam and Weinberg, 2013), re- static potential (Jolly et al., 2015; Lu et al., 2013; Tian et al., 2013;
flecting the flexibility of these cells to induce or reverse the
EMT process (Rosano` et al., 2006). In physics and chemistry,
the metastable state denotes an isolated system that is main-
tained in a delicate equilibrium and not in a state of least energy
(Cheng and Keller, 1998). Thus, metastability suggests a dy-
namic phase transition along the energy gradient. Adapting
this concept of metastability to EMT probably fails to accurately
reflect thermodynamic laws. Direct evidence of the energy tran-
sition that occurs during the EMT process is limited, although a
22 Cell 166, June 30, 2016
Figure 1. Transitions through the Different
States along the EMT Spectrum
EMT can be considered as a continuum, whereby
cells exhibit epithelial (E), intermediate (EM), and
mesenchymal (M) phenotypes. As cells make the
transition from left to right, they sequentially lose
apico-basal polarity and cell-cell adhesions and
they gain front-back polarity and enhanced cell-
matrix interactions. The colored spectrum de-
notes hypothetical transitions (x axis). Different
models predict that both stability and metastability
could occur during the phase transitions where
there is a point of low energy or a net thermody-
namic equilibrium (y axis), in function of the
intrinsic, multi-parametric EMT regulators (z axis).
These regulators include transcription factors
(SNAI1/2, ZEB, TWIST1, GRHL2, OVOL1/2, and
PRRX1), post-transcriptional regulators (miRNAs),
and the proposed epigenetic controls at the pro-
moters of epithelial genes (Tam and Weinberg,
2013). When the system is perturbed in response
to EMT signals (e.g., hypoxia, growth factors, etc.),
cells could pass through a thermodynamic hump
that leads to a distinct phenotype (e.g., EM1;
EM3). The backward arrows represent reversal to
more epithelial states. It is worth noting that the
existence of discrete metastable states (e.g., EM1,
EM3) has not been proven and that the existence
of quasi-stable transitional states is also predicted
and found in different contexts, including fibrosis
(e.g., EM2). Importantly, although full reversibility
is evident during embryonic development, it is not
clear whether there is a point of no return in the
EMT undertaken by adult cells. Finally, it seems
that, although the mesenchymal state prevents
metastatic outgrowth, a full reversion to the
epithelial phenotype is not required for metastatic
colonization. TJ, tight junction; AJ, adherens
junction; DS, desmosome.
linear energy gradient based on epigenetic changes between
different EMT states has been hypothesized (Figure 1) (Tam
and Weinberg, 2013). More recently, the free-energy landscape
of EMT was modeled, proposing three thermodynamically and
kinetically stable states for EMT: the epithelial, maturation, and
mesenchymal states (Zadran et al., 2014). Energy is released af-
ter exiting the epithelial state as the cell moves to a maturation
state, which corresponds to an intermediate EMT state (Zadran
et al., 2014), and this energy is then consumed for the cell to
reach the mesenchymal state. Thus, the intermediate EMT state
in this model is not equivalent to the metastable state described
with biophysical models. Computational modeling, including
those that consider the mutual inhibitory loops between several
microRNAs (miRNAs) and EMT transcriptional drivers like Snail1
and Zeb1, also accepts an intermediate hybrid EMT state that
could favor the progress of developmental programs and meta-
Zhang et al., 2014). The inclusion of additional reciprocal inhibi-
tory loops that involve other transcription factors (e.g., Zeb1 with
Ovol2 and Grhl2) and the description of these as phenotypic sta-
bility factors indicates that the network is capable of generating
additional intermediate stabilized states that, therefore, are not
necessarily metastable (Hong et al., 2015; Jolly et al., 2016).
Figure 1 depicts putative intermediate states in a hypothetical
diagram to highlight the transitional nature of the process, which
can include stable and metastable states.

Evidence of transitional EMT states comes from multiple con-
texts. Whereas many studies use the co-expression of epithelial
and mesenchymal markers to define the hybrid state (Huang
et al., 2013; Yu et al., 2013), cells do not need to gain mesen-
chymal traits in a partial EMT. Indeed, early EMT might only
involve a dampening of epithelial properties like apico-basal
polarity and a remodeling of junctional complexes in favor of
cell-substrate adhesions (Huang et al., 2012). Even when a
mesenchymal trait is gained, there is still much heterogeneity
in terms of the mesenchymal characteristic induced. In an anal-
ysis of 43 ovarian cancer cell lines, only 50% of cells with an in-
termediate phenotype (co-expressing cytokeratin and vimentin)
induced N-cadherin (Huang et al., 2013). This heterogeneity may
reflect different biological consequences of the intermediate
states, such that cells losing E-cadherin that do not gain N-cad-
herin will most likely behave distinctly during migration and inva-
sion to those that do gain N-cadherin, given their different adhe-
sive behavior (Chu et al., 2006; Halbleib and Nelson, 2006).
It is also worth noting that the partial EMT seems to be a final
state in some cases, as described during organ fibrosis. Dedif-
ferentiated renal epithelial cells do not seem to undergo a full
EMT nor do they undergo reversal during the course of the dis-
ease. Importantly, they do not engage in the invasion program
but rather they remain integrated in the tubules (Grande et al.,
2015). Hence, it is important to consider not only epithelial or
mesenchymal traits during epithelial transitions but also other
programs associated with EMT, such as invasion, increased
survival or decreased proliferation.
In sum, intermediate states probably reflect the delicate
balance of transcriptional drivers and suppressors of EMT.
This equilibrium is inevitably affected by epigenetic changes
(Tam and Weinberg, 2013) and by the main effectors in the cyto-
Figure 2. The Core Regulatory Machinery
of EMT
Although multiple non-coding RNAs control EMT,
two regulatory networks have been described
that can be considered as the core regulatory
machinery: the miR34-SNAI1 and miR200-ZEB1
axes. These two miR-transcription factor (TF) axes
employ a double-negative feedback mechanism
in which miR34-SNAI1 and miR200-ZEB1 repress
each other. SNAI1 and ZEB1 further repress
miR200 and miR34, respectively. At this level,
other TFs, such OVOL1/2 and GRHL2, further feed
into this core regulatory machinery to protect
the epithelial phenotype. The miR34-SNAI1 and
miR200-ZEB1 core not only contributes to the
epigenetic control of EMT, but are also targets
for epigenetic modifications. Downstream of the
miR34-SNAI1 and miR200-ZEB1 axes, regulation
of transcript processing would shape the land-
scape of epithelial and mesenchymal effectors,
for example, through alternative splicing. These
EMT effectors may also be subject to epigenetic
modifications. How other TFs, like TWIST1 and
PRRX1, affect the network and its epigenetic
control requires further study, although it is clear
that, unlike Snail and Zeb, they are much more
potent mesenchymal promoters than epithelial
repressors.
skeleton, the cellular machinery driving migration and invasion.
This wider view of EMT offers a more dynamic interpretation of
the fluidity and plasticity of this phenomenon (Figure 1).
Complex Regulatory Networks of EMT: Beyond
Transcriptional Control
EMT is modulated at different levels by integrating epigenetic
modifications, transcriptional control, alternative splicing, pro-
tein stability, and subcellular localization. Thus, the mechanisms
that govern the intermediary phases of EMT are non-linear.
Despite the efforts to identify common regulatory networks in
normal development and disease, it is clear that some pathways
may be unique to a given EMT event in a particular tissue or
tumor subset. The regulation of classical EMT centers on the
transcriptional suppression of the prototypic adhesion molecule,
E-cadherin (Lamouille et al., 2014; Thiery et al., 2009). The
activities of major EMT-TFs, such as SNAI1, SNAI2, ZEB1,
ZEB1, and TWIST1, have been described and reviewed exten-
sively (Lamouille et al., 2014; Peinado et al., 2007). With these
EMT-TFs taking center stage over the past decade, our under-
standing of EMT has evolved to explain how their transcripts
are expressed and processed by miRNAs (Lamouille et al.,
2013) or alternative splicing (Warzecha and Carstens, 2012).
Multiple miRNAs are thought to govern EMT, as reviewed else-
where (Daz-Lopez et al., 2014). As a prototypic regulatory
model, we focus here on the more elaborate networks involving
miR-200 and miR-34, together with ZEB1 and SNAI1. This regu-
latory network (Figure 2) establishes a negative feedback that is
thought to maintain epithelial homeostasis under normal condi-
tions (Puisieux et al., 2014). Indeed, various epithelial markers
are downstream targets of this regulatory loop, including E-cad-
herin, claudins, and occludins (Lamouille et al., 2014). Alternative
Cell 166, June 30, 2016 23
splicing mediated by epithelial-specific regulatory protein 1 and
2 (ESRP1 and ESRP2) also affects the maintenance of epithelial
features (Warzecha and Carstens, 2012). A panel of transcripts
involved in cell-cell adhesion, cell motility, and cell-matrix adhe-
sion are regulated by these ESRPs, including fibroblast growth
factor receptor 2 (FGFR2), p120-catenin, CD44, and Mena (War-
zecha and Carstens, 2012). Conversely, splicing programs pro-
moted by Quaking (QKI), SRSF2, and RBFOX2 are upregulated
during EMT (Bonomi et al., 2013; Braeutigam et al., 2014;
Conn et al., 2015). QKI promotes the generation of circular
RNAs (circRNAs) by non-sequential back-splicing of pre-
mRNAs, and RBFOX2 has multiple targets that include the re-
ceptor tyrosine kinase Ron, cortactin, and dynamin (Figure 2).
Therefore, the maintenance or loss of epithelial features is gov-
erned by how transcripts are processed to their mature forms
prior to translation. This systems view provides an alternative
way to describe the EMT spectrum in terms of regulatory net-
works, both quantitatively and qualitatively.
Importantly, the miRNA-TF regulatory circuits are not linear in
EMT, as there is substantial diversity in the regulation exerted by
different miRNAs in EMT. Within the miR-200 family, miR-200a/
b/c, miR-141, and miR-429 all target ZEB1, yet they probably
inhibit ZEB1 translation to a different extent. It is unclear how
other novel TFs recently implicated in EMT or MET affect the
interaction between the miR34/SNAIL and miR200/ZEB regula-
tory circuits, such as PRRX1 (Ocana et al., 2012), grainyhead-
like 2 (GRHL2) (Cieply et al., 2012), or connective tissue growth
factor (CTGF) (Chang et al., 2013). GRHL2 and OVOL1/2 form
a double-negative feedback loop with ZEB1 (Figure 2) (Hong
et al., 2015), and GRHL2 directly regulates the transcription of
miR-200 and OVOL1/2 upstream of ZEB1 (Chung et al., 2016;
Cieply et al., 2012). PRRX1 induces EMT in a SNAI1-independent
manner, yet cooperates with TWIST1 to drive invasiveness
(Ocana et al., 2012). Both PRRX1 and TWIST1 are more potent
mesenchymal inducers than epithelial repressors, and SNAI1
and ZEB1 are strong epithelial repressors and weaker mesen-
chymal promoters. In contrast to SNAIL and ZEB family mem-
bers, there is still little information regarding the regulation of
TWIST and PRRX by miRNAs or by other non-transcriptional
mechanisms (Figure 2).
Upstream and downstream epigenetic controls largely affect
the EMT miRNA-SNAIL-ZEB networks, as these EMT transcrip-
tional drivers are involved in a complex epigenetic regulatory
loop (Bedi et al., 2014). Interestingly, their regulation might
involve transitions through intermediate states (Jordan et al.,
2011). SNAI1 recruits chromatin modifiers, including SIN3A,
histone deacetylase 1 (HDAC1), HDAC2 (Peinado et al., 2004),
lysine-specific demethylase1 (LSD1) (Lin et al., 2010), compo-
nents of the polycomb repressor-2 (PRC2) complex (Herranz
et al., 2008), and the G9a and Suv39H1 histone methyltrans-
ferases (Dong et al., 2013; Lin et al., 2014). SNAI1 binds tran-
siently to its target promoters, triggering both transient and
long-lasting chromatin changes (Javaid et al., 2013). In addition,
SNAI1 can interact with a lysyl oxidase, LOXL2 (Peinado et al.,
2005), and regulate heterochromatin transcription (Millanes-Ro-
mero et al., 2013). On the other hand, ZEB1 represses its target
genes by recruiting the LSD1-containing co-repressor complex
(Wang et al., 2007), as well as HDAC1 and HDAC2 (Aghdassi
24 Cell 166, June 30, 2016
et al., 2012). ZEB proteins are required for the co-repression
in Jurkat cells mediated by a histone acetyltransferase (HAT)
domain-containing protein, Tip60 (Hlubek et al., 2001), and
ZEB1 itself is affected by the recruitment of PRC2 by the histone
methyltransferase, PRDM14 (Chan et al., 2013). Both inactive
(H3K27Me3) and active (H3K4Me3) histone marks can be found
at the ZEB1 promoter region, presumably to facilitate a switch
during EMT (Chaffer et al., 2013; Spaderna et al., 2008). There-
fore, the consequences of the epigenetic changes initiated by
these EMT-TFs during EMT are expected to confer dynamic
control over chromatin and nuclear organization during the tran-
scription of downstream EMT effector genes, thereby contrib-
uting to the plasticity evident during transitions.
The miR-200 family is also subjected to epigenetic modifica-
tions. The expression of miR-200 family members is regulated
by a histone demethylase, KDM5B (Enkhbaatar et al., 2013).
The miR-200b-200a-429 cluster is regulated by histone modifi-
cations mediated by the polycomb group and the EMT suppres-
sor GRHL2, and the miR-200c-141 cluster is regulated by DNA
methylation (Lim et al., 2013; Chung et al., 2016). This epigenetic
regulation of these EMT miRNA-TF networks further complicates
the entire regulatory landscape of EMT, especially given that
many EMT effector genes can also be directly regulated by his-
tone modifications and methylation, including E-cadherin
(Figure 2) (Wu et al., 2012). Histone acetylation is altered in
epithelial, intermediate, and mesenchymal trophoblast stem
cells, and these changes have been correlated with the expres-
sion of different TF combinations (Abell et al., 2011; Jordan et al.,
2011). Accordingly, an EMT acetylome could be derived by
crossing these changes in acetylation with known EMT signa-
tures (Jordan et al., 2011).
At the protein level, EMT is also regulated by post-translational
modifications including the ubiquitin-mediated degradation of
important effectors, such as that following the phosphorylation
of Snail1 and Twist1 by GSK3b or MAP kinases, respectively
(Hong et al., 2011; Zhou et al., 2004). F-box proteins also target
these and other EMT-TFs to the proteasome (Diaz and de Her-
reros, 2016). Phosphorylation also impinges into Snail1 subcellu-
lar localization (Du et al., 2010; Zhang et al., 2012a), in which nu-
clear import and export pathways have also been characterized
(Domnguez et al., 2003; Mingot et al., 2009, 2013). As such, it
seems to be a daunting task to construct a systems landscape
that encompasses the regulatory networks involving miRNA-TF
loops, transcript processing, and the epigenetic control of pro-
tein stability to determine what controls the continuous transi-
tions through the complete EMT spectrum. However, the recent
demonstration that a generic EMT signature is common to
various cancers (Tan et al., 2014) and other advances in our un-
derstanding of EMT in physiological and pathological conditions
makes it reasonable to believe that such broad systems analyses
are not so far away.
New Insights for EMT in Development
EMT drives important aspects of embryonic development. This
phenomenon became apparent to developmental biologists in
the late nineteenth century, presenting clear histological evi-
dence that, during gastrulation, mesenchymal cells originated
from the adjacent epiblast. EMT was established experimentally
by showing that embryonic and adult epithelia embedded in 3D
collagen gels converted into migratory, invasive fibroblast-like
cells (Greenburg and Hay, 1982). This seminal study provided
evidence that, contrary to popular belief, embryonic and also
adult epithelial tissues were less stable than expected. Different
model systems were explored to define the molecular mecha-
nisms driving this remarkable conversion. One important finding
was that rapid changes in epithelial cell shape were driven by the
complementary modulation of cell-cell adhesion and cell-sub-
strate interactions, mediated by N-CAM and E-cadherin, as
well as extracellular matrix (ECM) proteins such as fibronectin
and integrin receptors, respectively (Boucaut et al., 1984; Edel-
man et al., 1983; Takeichi, 1988). Another landmark was the dis-
covery that an ortholog of the Drosophila snail gene, expressed
during the formation of the ventral furrow (Leptin and Grunewald,
1990), was critical to the formation of the primary mesenchyme
during gastrulation in the chicken embryo (Nieto et al., 1994).
The first signal-transduction pathways leading to EMT were
then defined from genetic studies in Drosophila and in vertebrate
embryos. Here, we integrate some recent findings on EMT in
development with the knowledge accumulated over the past
two decades.
Gastrulation
Genetic studies in Drosophila revealed how dorsoventral polar-
ity, mediated by Toll receptor activation, engages a program
orchestrated by Twist and Snail. This program involves cell-cycle
repression by Tribbles, and the activation of actomyosin
contractility by T48 and Fog through RhoGEF2. EMT of the
invaginated epithelium in the ventral furrow is then promoted
by the activation of heartless, an FGFR tyrosine kinase (Lim
and Thiery, 2012). Snail promotes an E- to N-cadherin switch
(Oda et al., 1998) that, in conjunction with FGF signaling medi-
ated by heartless activation, initiates EMT in the nascent fly
mesoderm. Recent findings question whether the repression of
E-cadherin during the invagination process and/or the gain of
N-cadherin are in fact key events during fly gastrulation (Schafer
et al., 2014), with the suggestion that other mechanisms control
the changes in morphology and the individualization of the cells
in the mesoderm. One possibility to reconcile earlier data with
these findings could be that E-cadherin turnover is accentuated
upon forced overexpression in order to reduce the strength of
intercellular adhesion, giving rise to a much weaker phenotype
than that anticipated. Clearly, more work is required to clarify
how this intricate network is autoregulated when challenged,
leading to unpredictable and weak phenotypes and emphasizing
the robustness of gastrulation in Drosophila.
The analysis of the gene regulatory networks in sea urchin
gastrulation also indicated that Snail and Twist execute the
EMT process in primary mesenchyme formation (Saunders and
McClay, 2014). However, a detailed analysis of blastula epithelial
cell behavior defined five distinct sub-circuits involved in EMT:
basement membrane remodeling, motility, apical constriction,
loss of apico-basal polarity, and changes in intercellular adhe-
sion. Thirteen transcription factors contribute to the execution
of EMT, including Snail and Twist, but no single transcription
factor is expressed in all five sub-circuits, highlighting the
complexity and robustness of the system even in sea urchin
(Saunders and McClay, 2014). These findings are consistent
with the phenotype observed in mouse embryos, where Snail1
mutant embryos do not survive gastrulation (Carver et al.,
2001). However, the specific conditional deletion of Snail1 in
the epiblast affects mesoderm migration, but not its initial forma-
tion (Lomel et al., 2009; Murray and Gridley, 2006).
Strong gastrulation defects are observed in FGFR1 knockout
mice (Ciruna and Rossant, 2001). FGF signaling, together with
Nodal and Wnt3 signaling, is required for the generation of
migratory mesenchymal mesodermal cells (Lim and Thiery,
2012). FGF signaling maintains Snail1 expression, and Snail1
and Sox2/3 mutually repress each other, with Sox3 preventing
epithelial cell ingression and Snail1 promoting it by inhibiting
Sox3 in the primitive streak in chick and mouse embryos (Aclo-
que et al., 2011).
The winged helix-turn-helix Ets2 transcription factor is ex-
pressed in extraembryonic ectoderm trophoblasts, and it plays
a role in the formation and elongation of the primitive streak in
mouse embryos (Polydorou and Georgiades, 2013). Indeed,
Ets2 promotes the expression of Elf5 and Cdx2 in order to main-
tain trophoblast stemness, and it also induces bone morphoge-
netic protein 4 (BMP4) expression, which acts on the epiblast in a
paracrine manner to induce Wnt3. Wnt3 elevates Nodal and in-
duces Snail1, promoting primitive streak elongation and EMT,
respectively. These findings add another layer of complexity to
EMT signaling in mouse gastrulation.
Neural Crest and Mesectoderm
At the neurula stage, a group of stem/progenitor cells appears at
the dorsal border of the neural plate of vertebrates, the neural
crest (NC) (Baggiolini et al., 2015; Buitrago-Delgado et al.,
2015), which generates glial cells, most neurons in the peripheral
nervous system, some endocrine and paraendocrine cells, and
all melanocytes. NC cells share intercellular adhesion systems
with adjacent neural epithelial cells that are destined to give
rise to the CNS, yet they engage in EMT to form a population
of migratory cells that populate distant territories. The mecha-
nisms driving EMT induction in NC cells are only partially shared
across species, although a generic gene regulatory network has
been established (Simoes-Costa and Bronner, 2015), whereby
the EMT signaling module is believed to be embedded in a
rather complex network. Multiple feed-back loops regulate NC
development and guarantee the robustness of the system,
anticipating compensatory regulatory mechanisms. Epigenetic
regulation also occurs during NC specification and migration,
controlling the expression of important EMT regulators (Hu
et al., 2014).
In the chick embryo, the NC territory at the trunk level is
induced by reciprocal rostro-caudal gradients of retinoic acid
(RA) and FGF. The territorys borders are refined by downstream
signaling that involves opposing gradients of BMP4/noggin and
Wnt1, as well as epigenetic controls that establish poised chro-
matin domains of genes like Snail, which are activated before cell
emigration. In the NC territory, BMP4 induces N-cadherin cleav-
age by ADAM10 (Shoval et al., 2007), and it creates two
opposing gradients to control the downregulation of N-cadherin
and the expression of cadherin-6 (Park and Gumbiner, 2010),
events that transiently mark the territory from which the NC dis-
sociates. Cadherin-6 is also cleaved by ADAM10 and g-secre-
tase (Schiffmacher et al., 2014), and thus, it is downregulated
Cell 166, June 30, 2016 25
following crest cell emigration when these cells acquire cad-
herin-7. In zebrafish, cadherin-6 acts autonomously in NC pro-
genitors to localize activated RhoA in the apical domain. This
restricted distribution promotes actomyosin contractility and
constriction of the apical domain, releasing the apical junctional
complexes of NC cells to allow them to emigrate from the neural
fold (Clay and Halloran, 2014). In summary, the loss of apico-
basal polarity, the modulation of cadherins and other adhesion
molecules, and the profound remodeling of the actin cytoskel-
eton allow EMT to occur and cells to acquire a mesenchymal
migratory morphology.
At cranial levels, mesectodermal cells can also give rise
to mesodermal derivatives that include bone, cartilage, and
meninges. Believed to be part of the NC, recent findingsstill
subjected to debatepropose that this may be a distinct yet
adjacent ectodermal cell population that also undergoes EMT.
These cells would downregulate E-cadherin rather than N-cad-
herin as they dissociate from the non-neural epithelium of the
neural fold (Lee et al., 2013; Weston and Thiery, 2015). However,
in Xenopus embryos, migratory cranial cells still express N-cad-
herin during migration as they pursue collective cell migration,
having undergone only a partial EMT (Scarpa et al., 2015).
Heart
The heart develops from a group of mesodermal mesenchymal
cells during gastrulation that assembles as two cardiogenic
mesodermal layers (the primary and secondary heart fields),
completing a first round of EMT and MET. The second round is
activated in the splanchnopleuric mesoderm to form the inner
endothelial layer, and it is followed by a third round to form the
cardiac valves that arise from the dissociation of endothelial cells
in the atrio-ventricular region and outflow track (OFT). This third
round should perhaps more precisely be termed an endothelial-
mesenchymal transition (EndMT).
Quite distinct mechanisms drive the successive rounds of
EMT/EndMT and MET during heart development (Lim and
Thiery, 2012; von Gise and Pu, 2012). Whereas the first round
is basically a subprogram of gastrulation that depends on Wnt,
FGF, and TGFb signaling, the third is initially driven by the
BMP2 produced by myocardial cells. Nevertheless, BMP null-
homozygotes do not exhibit valve defects, indicating that
compensatory mechanisms exist similar to those seen in gastru-
lation and NC development. However, altering downstream ca-
nonical signaling, such as interfering with Smad4, leads to a
loss of the cardiac cushion. BMP2 can drive local EMT in the
atrioventricular region through the activity of its downstream
target Tbx2. The atrioventricular endocardium responds to the
myocardium through secreted BMP2 and TGFb1/2, triggering
canonical Smad pathways. All four ErbB receptors are important
for cardiac valve formation, and while ErbB2-ErbB3 hetero-
dimers are activated by neuregulin in the endocardium in an au-
tocrine manner (Lim and Thiery, 2012), ErbB3 signaling is also
evident in the myocardium and is driven by the hyaluronic acid
produced by hyaluronan synthase (HAS)-2, a Tbx2 target. Notch
signaling is another critical pathway activated in the endocar-
dium (Luxan et al., 2016), and all three pathways, Notch, BMP/
TGFb, and ErbB, contribute to endocardial EndMT to establish
the valve interstitial cells (Moskowitz et al., 2011; von Gise and
Pu, 2012).
26 Cell 166, June 30, 2016
The OFT obeys the same controls in the formation of the
conotruncal endocardial cushion, albeit with the additional
complexity that the cells arise from the second heart field and
the NC. EndMT operates under the Tbx2-BMP2 axis, and
when deregulated, retinoic acid (RA) can directly target Tbx2
transcription and reduce BMP2 levels. Indeed, ectopic RA
causes defects in the OFT cushion (Sakabe et al., 2012).
Mesothelial cells of the sinus venosus located at the posterior
end of the embryonic heart form the proepicardium anlage.
Bmp2 expression in this anlage is weak, inducing the expression
of Wilms tumor gene 1 (Wt1) and Tbx18 to sustain epicardial
identity while the cells migrate to the heart primordium to form
the primitive epicardium. Pre-epicardial cells migrate and
assemble as a single-layered epithelial sheet around the
myocardium through a MET-like process. Subsequently, the
epicardial layer undergoes EMT and gives rise to resident inter-
stitial fibroblasts within the myocardium, as well as smooth
muscle cells in the coronary arteries and at least 20% of the
endothelium (Cano et al., 2016). This process has recently
been considered as an extended process of the EMT of meso-
thelial cells, as the primitive epicardium never undergoes a full
MET (Ariza et al., 2016). Epicardial EMT is controlled by the
neurofibromatosis type 1 (NF1) gene, as its deletion induces pre-
mature and more extensive EMT in the epicardium (Baek and
Tallquist, 2012). Wt1 is also essential for this process, inducing
the expression of either Snail1 or b-catenin to promote cellular
dissociation and migration in the subepicardial layer. The
basic-loop-helix TCF21 also influences the EMT and lineage
specification of epicardial cells (Acharya et al., 2012). On the
other hand, the Notch pathway promotes EMT by activating
platelet-derived growth factor receptor (PDGFR) and producing
TGFb in cooperation with Wt1, which can induce RA production
by the Raldh2 enzyme (Lim and Thiery, 2012; Perez-Pomares
and de la Pompa, 2011).
Together, these findings show that proper heart development
requires a plethora of growth factors and pathways to drive
epicardium-myocardium interactions, which are still to be
comprehensively integrated (as required in other developmental
processes). It is widely accepted that pathways converge on the
activation of different combinations of EMT-TFs and that an
EMT code determines cell behavior, producing heterogeneity
and tissue specificity. Establishing such gene regulatory net-
works and feedback loops ensures the robustness of pivotal
developmental processes, as observed during gastrulation and
NC development.
Although EMT in development has inspired cancer re-
searchers to identify similar mechanisms in the progression of
carcinomas, it is now clear that the regulation observed in normal
development would not only be used, but also expanded on in
cancer, and that additional effort is needed to describe the
signaling networks that drive invasion and metastasis.
Protection of the Epithelial Phenotype
Despite the high degree of epithelial plasticity observed during
embryonic development, it is appropriate to stress that, in
normal adult epithelial tissues, various mechanisms guarantee
epithelial homeostasis and protect the epithelial phenotype in or-
der to maintain tissue integrity and organ function. In addition to
the aforementioned global program driven by epithelial-specific,
ESRP-mediated splicing (Warzecha and Carstens, 2012), the
epithelial-specific H2BK5Ac epigenetic mark also exists (Abell
et al., 2011). Moreover, Ovol zinc-finger TFs appear to act as pro-
tectors of the epithelial phenotype, with Ovol1/Ovol2 inhibiting
the cells capacity to engage into EMT by activating epithelial
genes and repressing multiple EMT drivers and mesenchymal
genes (Lee et al., 2014; Kitazawa et al., 2016). Ovol2 deletion
dramatically augments the EMT phenotype in epithelial cells of
the mammary gland, provoking considerable structural pertur-
bation during extension of the terminal end buds (Watanabe
et al., 2014). Elf5 shares similar properties in protecting the
epithelial state by suppressing Snail2 during mammary gland
development and metastasis (Chakrabarti et al., 2012).
New functions for p53 extend its role of guardian of the
genome (Lane, 1992) to include guardian of the epithelial in N-acetylglucosaminyltransferase V (GnT-V) transgenic mice
phenotype, as p53 also safeguards epithelial homeostasis by
directly activating the miR-200 family. In mammary epithelial
cells, the loss of p53 causes a decrease in miR-200c thereby
increasing EMT, with a concomitant increase in the stem cell pop-
ulation (Chang et al., 2011). The latter is in part mediated by the
upregulation of Bmi1 (Shimono et al., 2009), which is expressed
by cisplatin-resistant bladder cancer cells that behave as cancer
stem cell (CSC)-like cells, undergoing EMT with the potential to
form spheres (Zhang et al., 2012b). EMT is also prevented by
miR-200b, which inhibits mammosphere formation through tar-
geting Suz12, a subunit of the polycomb PRC2 complex (Iliopou-
los et al., 2010). Another PRC2 component, EZH2, supresses
EMT, invasion, and metastasis by repressing Snail1 in colorectal
cancer cells (Wang et al., 2015). Other protectors of the epithelial
phenotype include proteins associated with DNA damage repair,
such as the ubiquitin ligases FBXW7 and Siah, which prevent
EMT by targeting Zeb1 for degradation (Chen et al., 2015; Yang
et al., 2015), as well as ataxia-telangiectasia-mutated (ATM) ki-
nase, which represses Sox9 expression (Russell et al., 2015). In
some cell contexts, Sox9 behaves as an important transcription
factor associated with EMT and stemness.
The programs implemented to protect the epithelial pheno-
type are counterbalanced by other programs that promote
EMT. As such, epigenetically driven global reprogramming of
specific chromatin domains and specific splicing programs
occur during the induction of EMT (Bonomi et al., 2013; Braeuti-
gam et al., 2014; McDonald et al., 2011). While the final response
would depend on the relative contribution of these positive and
negative EMT programs, in the healthy adult, the protection of
epithelial homeostasis will generally prevail. Nevertheless, the
remarkable epithelial plasticity observed under physiological
conditions in certain tissues is worth noting, such as in the mam-
mary gland during puberty and pregnancy. Ductal elongation of
the mammary gland during puberty is driven by terminal end
buds. These multi-layered structures exhibit significant epithelial
cell plasticity to allow fat pad invasion through the collective
migration of cells with an intermediate EMT phenotype (Kurley
et al., 2012; Shamir and Ewald, 2015). As the reactivation of
EMT programs in the adult go far beyond this process, the
following sections discuss the implication of EMT in wound heal-
ing, fibrosis, and cancer, with a focus on partial phenotypes in
terms of both physiology and pathology.
The Partial EMT Associated with Wound Healing
During epithelial wound healing, cells at the edge of the wound
move into damaged area to re-establish normal tissue architec-
ture in a process referred to as re-epithelialization. Since epithe-
lial cells are fairly immotile, they must undergo behavioral
changes to move as a coordinated group of cells and heal
the wound, ultimately reverting to their epithelial phenotype to
reconstitute epithelial sheet integrity. Both basal and suprabasal
cells are involved in the repair process that involves a partial
EMT (Shaw and Martin, 2016). The partial EMT is thought to
rely on Snail2 under the control of the epidermal growth factor
receptor (EGFR)-extracellular signal-regulated kinase 5 pathway
in mouse skin explants (Arnoux et al., 2008). As such, wound
healing is defective in adult mice deficient for Snail2 (Hudson
et al., 2009). EGFR signaling also promotes re-epithelialization
through the upregulation of Snail and Twist (Terao et al., 2011).
In addition, axon guidance molecules play an important role in
this partial transition during wound healing in the mouse skin.
As such, upregulation of EphrinB1 in the basal and suprabasal
cells downregulate components of the cell-cell adhesion com-
plexes, loosening the junctions to release tension and enable
cell movement (Nunan et al., 2015). In fly larvae, Netrin A controls
EMT in the squamous epithelium by downregulating Frazzled, a
member of the deleted in colorectal carcinoma (DCC) family of
surface receptors, which are known to stabilize adherens junc-
tions through the phosphorylation of moesin (Manhire-Heath
et al., 2013). These studies offer insight into epithelial breakdown
during EMT, which is important given that Netrin is overex-
pressed in various cancers, and it induces cancer cell prolifera-
tion and migration through the activation of YAP signaling (Qi
et al., 2015).
In the lung, wound healing is also associated with an EMT
phenotype, typified by a reduction in cell-cell junctions, cell flat-
tening, the acquisition of migratory properties, and the expres-
sion of vimentin. Yet, this transient phenotypic change is limited
to the basal cells of the airway and there is no evidence that
type II alveolar cells, which are responsible for the production
and secretion of surfactant to reduce surface tension, undergo
EMT or adopt mesenchymal features. By contrast, the club (or
clara) cells in the bronchiole airways appear to undergo a tran-
sient EMT program during the regeneration of the bronchiolar
epithelium. Club cells act as stem cells, and they can terminally
differentiate into ciliated cells during lung tissue repair (Vaughan
and Chapman, 2013).
Wound healing is often investigated using the dorsal closure
model in Drosophila, as EMT- and MET-like events are coordi-
nated during this process. During dorsal closure, epidermal
flanksamnioserosamigrate to extend over the gap in the
epidermis, and as the epidermis progressively expands on the
outer surface of the dorsal territory, the transient amnioserosa
epithelium becomes internalized. Epidermal pioneer cells at the
leading edge undergo a partial EMT, forming filopodia and
lamellipodia that actively promote cell migration through
Cdc42/Rac-mediated activation of actomyosin contractility.
The Scribble apico-basal polarity complex recruits p21-activated
kinase (PAK) to the leading edge for polarized cell migration, and
when closure is complete, PAK recruits the Scrib complex to
Cell 166, June 30, 2016 27
restore septate junctions and apico-basal polarity (Bahri et al.,
2010). GRH, the homolog of mammalian GRHL-1, -2 and -3, reg-
ulates wound healing in Drosophila amnioserosa through the
establishment of septate junctions (Narasimha et al., 2008).
GRHL3 is also involved in epithelial homeostasis in the mouse
(Ting et al., 2005), where it contributes to wound healing by acti-
vating transglutaminase 1 and RhoGEF19 (Darido and Jane,
2010). However, GRHL2 can compensate for the loss of
GRHL3 to heal the wounds of hind limb amputated mouse
embryos (Boglev et al., 2011), suggesting redundancy is at play.
It is important to note that, while EMT in cancer is deleterious,
wound-healing-driven EMT induced in response to injury is
beneficial. Therefore, analyzing the similarities and differences
between these two types of EMT will be essential to better
design specific therapies (Leopold et al., 2012). Nonetheless,
some EMT-healing responses may also be deleterious, such
as opacification of the posterior capsular (PCO), a major compli-
cation after surgery for cataract treatment (Xiao et al., 2015), or
the exaggerated healing responses that lead to fibrosis and scar-
ring. Interestingly, defective healing and increased scarring was
recently associated with prolonged inflammation (Qian et al.,
2016), highlighting the tight connection between a sustained
inflammatory response and the progression of fibrosis (as dis-
cussed below).
Reconciling the Role of EMT in Fibrosis
Fibrosis, or tissue scarring, is a hallmark of many chronic degen-
erative disorders and is associated with reduced organ function
and eventual organ failure. Fibrotic disease is on the increase; for
example, idiopathic pulmonary fibrosis (IPF) is estimated to
match the incidence of stomach, liver, testicular, and cervical
malignancies (Hutchinson et al., 2015). Fibrosis typically ensues
through the progressive accumulation of ECM deposited by my-
ofibroblasts. The origin of these myofibroblasts has been
debated for many years, with EMT being considered by some
authors to drive the transformation of epithelial and endothelial
cells into myofibroblasts (Iwano et al., 2002; Zeisberg et al.,
2007b), whereas lineage tracing in transgenic mice indicates
that the contribution of those cells to the population of myofibro-
blasts is negligible (Humphreys et al., 2010; Li et al., 2010a;
LeBleu et al., 2013). Recent advances in fibrosis-EMT research
has helped to shed light on some of these issues.
The EMT Debate in Renal Fibrosis May Be Explained by
Partial Activation of the Program
Early insight into the potential benefit of abrogating EMT in
fibrotic conditions came from the finding that mice lacking
Smad3 were protected against tubular interstitial fibrosis (TIF)
(Sato et al., 2003). Smad3 acts downstream of TGFb, one of
the main EMT inducers, and indeed, Smad2/3 activation medi-
ates renal fibrosis in response to TGFb and angiotensin II
(Yang et al., 2010a). Furthermore, reactivation of Snail and
EMT in renal epithelial cells can induce renal fibrosis and renal
failure (Boutet et al., 2006). However, there is no evidence of
the migratory potential of renal epithelial cells in vivo despite their
accumulation of mesenchymal markers when observed in cul-
ture upon treatment with TGFb (Humphreys et al., 2010; LeBleu
et al., 2013). New evidence may help to understand the mecha-
nisms behind renal fibrosis and resolve this controversy (Grande
28 Cell 166, June 30, 2016
et al., 2015; Lovisa et al., 2015). It seems that, although renal
epithelial cells do not directly generate myofibroblasts, deletion
of Twist or Snail in renal epithelial cells significantly attenuates
interstitial fibrosis in mouse models of TIF: unilateral ureteral
obstruction (UUO), folic acid administration (FA), or nephro-
toxic-serum-induced nephritis (NTN) (Grande et al., 2015; Lovisa
et al., 2015).
Yet, how can EMT-TFs be so relevant for the development of
fibrosis if kidney epithelial cells are not the source of myofibro-
blasts? These two recent studies both suggest that renal epithe-
lial cells undergo a partial EMT: one pointing to perturbations in
transporter proteins and cell-cycle regulation (Lovisa et al., 2015)
and the other showing that epithelial cells lose epithelial markers
and apico-basal polarity, but they remain integrated in the tu-
bules (Grande et al., 2015). Importantly, the injured epithelial
cells relay signals to the interstitium to promote the differentia-
tion of fibroblasts into myofibroblasts and the subsequent fibro-
genesis. The cells that underwent a partial EMT also secrete exo-
somes and cytokines that help recruit (1) bone-marrow-derived
mesenchymal cells that also differentiate into myofibroblasts to
promote fibrogenesis and (2) macrophages that sustain inflam-
mation (Borges et al., 2013; Grande et al., 2015; Lovisa et al.,
2015). Interestingly, although Snail factors were considered
purely as transcriptional repressors, Snail is converted into an
activator upon its binding to Twist (Rembold et al., 2014) and
also upon binding to CREB-binding protein (CBP). The latter al-
lows Snail1 to directly activate the transcription of inflammatory
cytokines genes in cancer cells and during fibrosis (Grande et al.,
2015; Hsu et al., 2014; Lovisa et al., 2015).
Thus, even though kidney tubular cells do not become
collagen-producing fibroblasts, they undergo a partial EMT,
and in a paracrine manner, they contribute significantly to fibro-
genesis and inflammation, two hallmarks of fibrosis (Figure 3).
Is There a Role for EMT in Liver and Lung Fibrosis?
Evidence to clarify the role of EMT in liver fibrosis has been less
forthcoming. Hepatocytes do not seem to undergo EMT in vivo,
and they are not the source of collagen-producing cells in liver
fibrosis (Taura et al., 2010). In fact, the transdifferentiation of he-
patic stellate cells are reportedly responsible for 90% of the
myofibroblast population in transgenic mice (Mederacke et al.,
2013), with neither hepatocytes nor cholangiocytes (the epithe-
lial cells of the bile duct) seeming to undergo EMT (Chu et al.,
2011; Scholten et al., 2010). These findings contrast with earlier
lineage-tracing studies showing that fibroblasts were derived
from hepatocytes that underwent EMT (Zeisberg et al.,
2007b). Others have reported that hepatic stellate cells secrete
collagen I to trigger EMT in epithelial hepatoma cells (Yang
et al., 2014), that fibrosis can be repressed by inhibiting TGFb
signaling (Park et al., 2015), and that miRNAs can modulate
EMT in cultured hepatocytes through TGFb inhibition (Brockhau-
sen et al., 2015; Zhao et al., 2014; Zhao et al., 2015). Importantly,
Snail deletion in hepatocytes attenuates liver fibrosis in adult
transgenic mice (Rowe et al., 2011), also suggesting that hepa-
tocyte-EMT may play a role in liver fibrosis.
In the light of these studies and the new data garnered from
renal fibrosis (Grande et al., 2015; Lovisa et al., 2015), it is
tempting to speculate that EMT contributes to the development
of liver fibrosis. In parallel with that observed in the kidney,

damaged hepatocytes could secrete signals after undergoing a
partial EMT, which promotes stellate cell transdifferentiation
and enhances inflammation. This would be consistent with
data obtained in mouse models of fibrosis in Snail-deficient he-
patocytes (Rowe et al., 2011). This partial EMT hypothesis is also
supported by findings in lung fibrosis, in which a subset of
epithelial cells in patients with IPF expresses both epithelial
and mesenchymal markers (Marmai et al., 2011).
Therapeutic Strategies in Fibrosis
As well as being a very potent EMT inducer, aberrant TGFb
signaling is the main cause of fibrosis, and it provokes a massive
accumulation of ECM. How or why TGFb signaling becomes
constitutive is unknown (Marmai et al., 2011), but nevertheless,
attenuating TGFb signaling has been the main strategy to fight
fibrosis (as in colorectal cancer, in which TGFb inhibitors
block the crosstalk between the stroma and cancer cells to
halt disease progression) (Calon et al., 2015). We describe
below different strategies proposed to inhibit the TGFb pathway
as a proof of concept of the benefits of targeting EMT, albeit
some targets may not fulfill the criteria for the optimal targeting
in patients.
Figure 3. EMT and Fibrosis
Following one of unilateral ureteral obstruction
(UUO), folic acid treatment (FA), or nephrotoxic
serum-induced nephritis (NTN), renal epithelial
cells undergo a partial EMT characterized by the
loss of epithelial and specific differentiation
markers and accompanied by the attenuation of
their proliferative capacity. These dedifferentiated
damaged renal epithelial cells persist in the tu-
bules, relaying instructive signals to the inter-
stitium to promote myofibroblast differentiation
and the recruitment of immune cells, thereby
promoting fibrogenesis and sustaining inflamma-
tion. All these effects are mainly due to the acti-
vation of EMT-TFs, including Snail and Twist, as
their specific deletion in renal epithelial cells
strongly attenuates the development of the
hallmarks of fibrosis (adapted from Grande et al.,
2015).
STAT3 is a downstream mediator of
TGFb signaling and induces EMT in coop-
eration with active K-Ras by increasing
Snail expression in cancer cells (Saitoh
et al., 2016). STAT3 is also reported to
be elevated in lung biopsies from patients
with IPF and in mice with fibrotic lungs.
Inhibiting STAT3 with a synthetic small-
molecule inhibitor (C-188-9) attenuates
fibrosis in bleomycin-treated mice (Pe-
droza et al., 2016). Agonists of another
endogenous inhibitor of TGFb signal-
ing, the orphan nuclear receptor 4A1
(NR4A1), can also inhibit fibrosis in skin,
liver, lung, and kidney (Palumbo-Zerr
et al., 2015), and this is consistent with
the inhibition of TGFb-induced EMT and
metastasis by the expression of NR4A1
itself (Zhou et al., 2014). Others have tar-
geted TGFb signaling in mouse models by modulating the activ-
ity of BMP components, including administration of BMP7 (Zeis-
berg et al., 2003) and the use of small peptides to specifically
inhibit the BMP-Alk3 receptor interaction (Sugimoto et al., 2012).
The prevention or reversal of fibrosis requires long-lasting sup-
pression and presumably, tissue remodeling. Epigenetic modifi-
cations can perpetuate fibroblast activation in renal fibrosis
(Bechtel et al., 2010) and, given that some epigenetic modifica-
tions can be reversible, epigenetic modulators such as miRNAs
are increasingly being targeted for the prevention or reversal of
fibrosis, although there are still concerns about the off-target
or toxic side effects associated with the delivery of miRNA inhib-
itors (Pottier et al., 2014). Examples of putative candidates are
miR-21 and miR-181, linked with EMT-driven fibrosis in epicar-
dial mesothelial cells (Brnnum et al., 2013), hepatic stellate
cells, and hepatocytes (Zhao et al., 2014; Brockhausen et al.,
2015). Other miRNAs have been proposed as beneficial against
fibrosis, after showing that they inhibit EMT: miR-200b and miR-
34c inhibit EMT in TIF (Morizane et al., 2014; Oba et al., 2010;
Tang et al., 2013); the miR-106b-25 cluster (miR-106b, miR-93,
and miR-25) is markedly downregulated in TGFb1-induced
Cell 166, June 30, 2016 29
EMT in renal HK2 cells (Tang et al., 2014); the loss of miR-101
promotes EMT and fibrosis in hepatocytes through TGFb activa-
tion (Zhao et al., 2015); and overexpression of let-7 miRNA in pri-
mary fibroblasts causes changes consistent with the inhibition of
EMT (Huleihel et al., 2014). These miRNAs could be considered
in anti-fibrotic therapeutic strategies that seek to restore their
function. As such, the intravenous injection of synthetic RNA du-
plexes that mimic miR-29 can recover its function and even
block pulmonary fibrosis in mice (Montgomery et al., 2014).
Heat shock proteins (HSPs), molecular chaperones that assist
in protein folding, are overexpressed in numerous cancers (Lia-
nos et al., 2015), and they have been linked with fibrosis (Bellaye
et al., 2014). HSP27 inhibition (OGX-427) destabilizes Snail and
inhibits TGFb-induced EMT and fibrosis (Wettstein et al.,
2013). In fact, blocking HSP27 with a synthetic tetrapeptide
(Ac-SDKP) or with the OGX-427 antisense oligonucleotide (an
anticancer agent in phase II clinical trials) can impair EMT
(Deng et al., 2014, Wettstein et al., 2013).
Fibrosis is associated with most cardiac diseases, leading to
stiffness of the cardiac tissue and dysfunctional cardiac contrac-
tion (Krenning et al., 2010). Patient survival rates are reported to
deteriorate as the proportion of fibroblasts rises (Gulati et al.,
2013). In the heart, a subset of endothelial cells undergo TGF-
b-mediated EndMT, and these fibroblasts are associated with
heart fibrosis (Zeisberg et al., 2007a). In addition, recent work
shows that approximately 35% of cardiac fibroblasts undergo
transdifferentiation into endothelial cells in a p53-dependent
manner through a mesenchymal to endothelial transition
(MEndoT), contributing to neovascularization after ischemia
(Ubil et al., 2014). Again, p53 could be a beneficial agent, and
reversion to the epithelial or endothelial phenotype should be
the preferred strategy in anti-fibrotic therapies.
In summary, the extensive exploration of EMT in fibrotic dis-
eases has helped identify therapeutic targets that predominantly
hinder TGFb signaling (Chen et al., 2013; Wang et al., 2013). How-
ever, inhibiting TGFb will also impede its beneficial effects (Mehal
et al., 2011) and it may therefore be more desirable to target
its principal downstream pro-fibrotic effectors (e.g., STAT3
or NR4A1), or alternatively, restore the expression of epithelial
miRNAs. These potential therapeutic targets can ameliorate
fibrosis by attenuating EMT and EndMT, offering an opportunity
to reverse fibrosis via MET. As such, inactivating Snail1 in mice
with established renal fibrosis improves organ morphology and
function, significantly attenuating the disease (Grande et al., 2015).
EMT in Cancer
EMT is thought to be activated in cancer cells, linked to their
dissociation from the primary tumor and their intravasation into
blood vessels (Thiery et al., 2009). However, the impact of the
EMT in cancer progression and patient survival is still far from
fully understood. From skepticism (Tarin et al., 2005) to funda-
mental support (Lamouille et al., 2014; Thiery et al., 2009; Ye
and Weinberg, 2015), more recent data suggest notes of caution
on the true role of EMT in cancer progression (Fischer et al.,
2015; Zheng et al., 2015). Nevertheless, studies in the last
decade have fueled the interest in EMT in the cancer field, mak-
ing it necessary to discuss recent advances regarding this com-
plex biological process.
30 Cell 166, June 30, 2016
Despite the vast body of literature regarding the role of EMT in
cancer, its applicability in cancer diagnosis and treatment has
remained limited, in part due to the intrinsic heterogeneity of
the actual tumor cells and tumor microenvironment when
compared with the relative homogeneity of cell culture models.
Thus, there are frequent debates as to whether EMT is truly rele-
vant to cancer in vivo. The execution of EMT in cancer is not ho-
mogeneous, lending further support to the need to view EMT as a
spectrum of intermediate states. Indeed, the description of the
tumor invasive front as being functionally distinct from the
main tumor bulk (Brabletz et al., 2001) points to the heteroge-
neous nature of the EMT program executed within the tumor
microenvironment. The tumor invasive front, in which the EMT
program is executed, has the hallmarks of a mesenchymal
phenotype, with a weakened cell adhesion system. By contrast,
the main tumor bulk remains largely epithelial (Figure 4). Thus,
there is probably an EMT gradient from full, (some focal events
at the tumor invasive front) to partial, to no (main tumor bulk)
EMT (Huang et al., 2013). Furthermore, this gradient may be
more or less steep in different tumors, reflecting the intrinsic
molecular heterogeneity arising from diverse driver mutation
profiles or altered signaling networks. For example, the circu-
lating tumor cells (CTCs) in breast cancers with a triple-negative
molecular subtype (ER/PR/HER2) tend to have a more mesen-
chymal phenotype. Therefore, the overall biology of the tumor
will likely dictate the frequency of identifying CTC subpopula-
tions with a partial or full EMT.
The tissue of origin may also influence the heterogeneity in
the execution of EMT. A quantitative EMT scoring system was
recently established based on gene expression profiles, ranking
the EMT state from 1.0 to +1.0 (Tan et al., 2014). This system
was used to highlight that the developmental lineage of each
cancer type is defined by both a micro- and macro-EMT
gradient, and each cancer type has a distinct propensity to
exhibit diverse EMT states. Expectedly, cancers that arise from
the mesoderm or NC lineages consistently show higher EMT
scores in both in vivo tumors and cultured cell lines. In these can-
cers (e.g., osteosarcoma and malignant melanoma), the range of
intrinsic EMT gradients is more restricted than in solid tumors of
epithelial origin, such as ovarian, breast, and lung carcinomas.
These different EMT gradients might help explain the inconclu-
sive clinical significance of EMT found in the literature. Given
the wide range of EMT states in ovarian, breast, and lung carci-
nomas, their clinical significance should be considered in
conjunction with their intrinsic molecular profiles, such as the
gene expression-based molecular subtype (GEMS) for ovarian
and breast cancers and the EGFR mutational status or ALK
fusion transcript status for lung cancer. In ovarian cancer, the
EMT status is closely linked to the reported GEMS, with worse
GEMS prognosis correlated with higher EMT scores (Tan et al.,
2013). Furthermore, the EMT status consistently predicts over-
all- (OS) and disease-free survival (DFS) (Tan et al., 2014). Collec-
tively, these findings offer a new perspective on the translational
applicability of EMT in prognosis.
In the absence of cell lineage analyses, the expression of
epithelial and mesenchymal markers in primary tumors is a
good indicator of a potential EMT. The existence of intermediate
phenotypes should also allow cells in the stromal compartment
Figure 4. The Metastatic Cascade
This scheme aims to illustrate the complexity of the metastatic cascade from the point of view of cell plasticity, from carcinoma cell heterogeneity in the primary
tumor to the different strategies that cancer cells adopt to survive in the bloodstream and colonize distant sites. EMT is a limited focal event in the primary tumor
that could occur upon the interaction of carcinoma cells with the microenvironment, in which cancer-associated fibroblasts (CAFs) and immune cells can be
found, including different types of natural killer T and B lymphocytes and inflammatory cells (not shown). The stroma also includes tumor-associated macro-
phages (TAMs) and other bone-marrow-derived cells. Some cells are possibly recruited prior to the establishment of primary and metastatic malignancies
(Barcellos-Hoff et al., 2013). CAFs are like myofibroblasts (Lau et al., 2016) and TAMs are M2-type macrophages (Hsu et al., 2014), indicating that the micro-
environment in the primary tumor is similar to that found in fibrosis, including an important inflammatory component. Other stromal cells also play a major role in
tumor progression (Gentles et al., 2015). As expected from a focal event, the proportion of carcinoma cells with EMT features in primary breast tumors does not
exceed 3% in estrogen receptor (ER)-positive tumors and 10% to 15% in ER-negative tumors. However, the majority of circulating tumor cells (CTCs) exhibits an
intermediate EMT phenotype. Cells that undergo EMT in the primary tumor can also help epithelial cancer cells to invade. CTCs may be derived from carcinoma
cells that undergo EMT in situ in the primary tumor or they may acquire intermediate EMT phenotypes in circulation, particularly when in clusters exposed to high
concentrations of TGFb originating from associated platelets. Recent data suggest that microemboli may be able to extravasate and that they exhibit higher
clonogenic potential (Aceto et al., 2014; Au et al., 2016). Cancer cells in metastatic outgrowths are clearly epithelial-like, and they can be identified morpho-
logically and molecularly as having been derived from the primary tumor, again highlighting the plasticity of tumor cells. This is also consistent with data showing
that mesenchymal cells must revert to the epithelial phenotype to colonize their new destination. The plasticity of carcinoma cells is therefore of upmost
importance to escape death during the different stages of tumor progression. Mesenchymal-like cells are better adapted to cell deformation, resisting shear
stress and being more resistant to drugs. Tumor dissemination in the lymph nodes is much less documented, albeit it is a major aspect of tumor staging (not
shown). Histopathological examination of proximal (sentinel) lymph nodes show either dispersed carcinoma single cells, micrometastases (<2 mm), or massive
invasion. The latter is compatible with findings indicating that lymph nodes can be colonized after collective cell migration (Giampieri et al., 2009).

(which maintain the epithelial markers of their tissue of origin) to
be identified. The distribution of a number of epithelial and
mesenchymal markers was first established in a breast cancer
tissue microarray (Sarrio et al., 2008). More recently, mesen-
chymal markers were found in 12% of the basal molecular sub-
types of breast carcinoma. This percentage is likely to be higher
in the claudin-low molecular subtype. Furthermore, cells ex-
pressing mesenchymal markers are also likely to exist among
epithelial carcinoma cells in ErbB2 and luminal molecular
subtypes; albeit, to a lesser extent (Tan et al., 2014; Yu et al.,
2013). It is also interesting to note that, in cancer, as in embryonic
development, EMT may be a focal rather than a global event,
probably a response of cancer cells to their local microenviron-
ment (Figure 4). Macrophages participate in EMT induction
within primary tumors in transgenic mouse models, orthotopic
xenografts (Wyckoff et al., 2004), and primary human breast car-
cinomas, interacting with carcinoma cells adjacent to the endo-
thelial cells (Robinson et al., 2009). The latter suggests that
macrophages may be also critical for local intravasation. M2a
macrophages migrate toward carcinoma aggregates to induce
the dissociation and migration of single carcinoma cells through
the local production of TGFb and direct ICAM-1-b2 integrin-
mediated cell-cell contact. Endothelial cells also contribute to
their dissociation through diffusible scatter factors, such as
HGF (Bai et al., 2015a; Bai et al., 2015b). Nevertheless, it is
crucial to consider the nature of CTCs as indicators of the next
step in the metastatic cascade.
EMT and Circulating Tumor Cells
Most CTCs express both epithelial and mesenchymal markers
(Khoo et al., 2015; Yu et al., 2013), suggesting that an active
EMT process is likely to operate during the dissemination of car-
cinoma cells (Figure 4) (Thiery and Lim, 2013). In addition, after
transient amplification of breast cancer CTCs, the cells exhibit
a full range of EMT phenotypes (Khoo et al., 2015), with evidence
indicating that CTCs express ECM components potentially
involved in the successful colonization of distant organs (Ting
et al., 2014).
A landmark study revealed that CTCs in blood from metastatic
breast cancer patients can serve to evaluate prognosis and
response to treatment (Cristofanilli et al., 2004), and this was
recently confirmed when CTCs quantified in metastatic patients
were shown to predict OS and progression-free survival (Bidard
et al., 2014). However, a recent clinical trial could not identify an
effective second-line chemotherapy strategy for patients whose
CTC numbers were not diminished by first-line chemotherapy
(Smerage et al., 2014). Clearly, there is an urgent need to under-
stand more about the molecular attributes of CTCs, particularly
how their presence and EMT status reflect treatment response

Cell 166, June 30, 2016 31

and malignant potential. Several studies have addressed the
value of CTC monitoring and phenotyping during neoadjuvant
and adjuvant therapies (Alix-Panabie`res and Pantel, 2014;
Khoo et al., 2015) and their use in drug sensitivity testing (Yu
et al., 2013). Importantly, cancer therapy affects CTC number
and phenotype, with refractory patients having more mesen-
chymal-like CTCs and patients who are responding to treatment
demonstrating significantly fewer CTCs with a more epithelial-
like phenotype (Yu et al., 2013). These findings are in agreement
with recent studies highlighting the importance of EMT in confer-
ring chemoresistance in breast and pancreatic cancer models
(Fischer et al., 2015; Zheng et al., 2015).
Intriguingly, the EMT program may be engaged at premalig-
nant stages, as shown in an experimental model of pancreatic
cancer, especially at sites of inflammation (Rhim et al., 2012).
This suggests that EMT may be a very early event in tumorigen-
esis and is consistent with the presence of disseminated tumor
cells (DTCs) at very early tumor stages in breast cancer patients
(Bidard et al., 2008; Husemann et al., 2008). CTCs are mainly
identified with the EpCAM antibody, which may exclude cells
that have undergone a more complete EMT and have lost this
epithelial marker. Thus, lineage tracing experiments in animal
models are required to identify CTCs in different states, and
this may also offer a way to explore whether CTCs only undergo
transitions after reaching the metastatic site or if MET can also
occur in the bloodstream. Interestingly, circulating tumor
clusters are more effective in colonizing secondary sites than
single mesenchymal CTCs (Aceto et al., 2014). These clusters
comprise distinct clonal carcinoma cells which are held together
through plakoglobin-mediated intercellular adhesion. Again, this
highlights the requirement for mesenchymal cancer cells to at
least partially reverse to the epithelial state for metastatic growth
(Nieto, 2013). CTC clusters formed through platelet interactions
are also correlated with a worse prognosis, and mesenchymal
CTCs may interact with epithelial-like carcinoma cells and
drag them along to secondary sites (Figure 4). It was unclear
how such aggregates can reach secondary sites through the
510-mm lumens of blood capillaries in the lung, but recent
data show that clusters of %20 cells can traverse these constric-
tions (Au et al., 2016). Extravasation is a very efficient process
and even untransformed epithelial mammary cells can reach
the lung after being injected into the bloodstream (Podsypanina
et al., 2008). If epithelial cells, and cell clusters, can efficiently
disseminate and extravasate, the EMT/MET pathway may
not be necessary in all cells for secondary tumor formation.
This directly impinges on the requirement for EMT in tumor
progression.
Is EMT an Essential Element of the Metastatic Cascade?
Most cancer EMT studies, including mechanistic analyses, are
conducted on cultured cells, and the vast majority of in vivo
studies are carried out after injection or xenografting of parental
or manipulated cancer cells, which are likely to show distinct
EMT features. Some mouse breast cancer reporter models
have been used to follow the endogenous activation of Snail
TFs (Tran et al., 2014; Ye et al., 2015), showing that Snail1 acti-
vation and endogenous EMT occurs in the primary tumor, and
that, although important for invasion and the formation of
CTCs, EMT is not required for metastatic colonization. These
32 Cell 166, June 30, 2016
data are consistent with the lack of metastatic outgrowth from
cells with constitutive mesenchymal traits, and it highlights the
transient nature of EMT and the cells need to revert to a more
epithelial phenotype for colonization (Ocana et al., 2012; Tsai
et al., 2012).
To better understand the contribution of EMT to metastatic
colonization, Fsp-1 or vimentin GFP-reporter lines were used
to monitor the expression of mesenchymal markers in a
MMTV-PyMT breast cancer model (Fischer et al., 2015). While
this provided evidence of EMT in a small proportion of cells in
the primary tumor, these GFP-positive cellseven though pro-
portionally enriched in the CTC populationdid not seem to
contribute significantly to the formation of metastatic out-
growths. Others show that pancreas-specific loss of either Snail
or Twist does not block systemic dissemination or the formation
of metastasis in pancreatic ductal adenoma carcinoma (PDAC)
(Zheng et al., 2015). Together, these findings prompted the au-
thors to suggest that EMT may be dispensable for carcinoma
progression to a metastatic state.
Although these data are provocative, EMT cannot yet be ruled
out as a driver of cancer progression (Maheswaran and Haber,
2015). First, the cooperation between the multiple EMT-TFs sug-
gests that loss of individual factors may be insufficient to prevent
cell invasion and dissemination, particularly in PDAC, in which
even normal cells do not exhibit a strong epithelial phenotype.
Furthermore, Fsp-1 is not a universal marker of EMT and it would
not be activated in the primary tumors of Neu and PyMT breast
cancer models (Trimboli et al., 2008), albeit EMT has been
described in both (Ye et al., 2015). Second, the few cells that un-
derwent EMT in the primary tumor (Fischer et al., 2015) are
consistent with the numbers observed in other studies (Tan
et al., 2014). Third, the relative enrichment of mesenchymal
markers in CTCs confirms their enhanced ability to invade and
intravasate. Fourth, the comparatively low contribution of the
vimentin-expressing cells to metastases calls for an analysis of
other markers that may better assess intermediate EMT pheno-
types and for the evaluation of a possible cooperation between
mesenchymal and epithelial cells (Tsuji et al., 2009). Indeed,
EMT may facilitate the invasion and intravasation of other cells
that retain their epithelial character (Figure 4). As such, even if
EMT were more limited than anticipated, it would still be required
for tumor progression to the metastatic state (Li and Kang, 2016).
A recent study using intravital microscopy in mice has detected a
pool of breast tumor cells that spontaneously undergo EMT,
become motile, and disseminate and then reverse to the epithe-
lial state upon metastatic outgrowth (Beerling et al., 2016). More
studies like these are needed in other types of cancer in order to
assess the contribution of EMT as a crucial requirement for the
progression of primary tumors toward the metastatic state, as
consistently suggested in different models and patient datasets.
EMT and Cancer Stem Cells
The induction of both EMT and stemness at the invasive fronts
of tumors was first suggested to explain how CSCs disseminate
and seed fully heterogeneous tumors at secondary sites (Bra-
bletz et al., 2005). Indeed, the EMT-derived stem cell phenotype
in the mammary glandcapable of forming mammospheres
following EMT induction and expressing a CD44highCD24low pro-
file (Mani et al., 2008; Morel et al., 2008)was reminiscent of that
of CSCs. This profile has since been identified in numerous CSC
subpopulations (Gupta et al., 2009a), with other groups reporting
high intracellular aldehyde dehydrogenase 1 (ALDH1) activity as
a marker of stemness (Ginestier et al., 2007). Recent indications
submit that CD44high and ALDH1 activity do not generally coexist
in CSCs (Liu et al., 2013a), suggesting the existence of different
CSC types. The gene signature identified for the induction of
pluripotency is also relevant to the persistence of CSCs, with
Oct4 and Nanog ectopically inducing CSC-like properties
and enhanced features of EMT (Kong et al., 2010). This is similar
to other genes involved in maintaining self-renewal capacity,
including b-catenin, Bmi1, Gli1, and Pou5f1. Within the
CD44highCD24low population of triple-negative breast cancers,
there are discrete epithelial and mesenchymal cell types that
are differentiated by their expression of a6-integrin splice vari-
antsa6A (epithelial) and a6B (mesenchymal)with the a6B-
b1-integrin noted as essential for CSC function, mammosphere
formation, and anchorage-independent growth (Goel et al.,
2014). Both a6-integrin and CD44 are alternatively spliced by
ESRPs, which as already discussed, protect the epithelial
phenotype and, hence, promote the presence of epithelial a6
and CD44 isoforms in these cells (Goel et al., 2014; Warzecha
et al., 2009). Accordingly, it would be interesting to correlate
the combinations of integrins and CD44 isoforms with ALDH1
activity in primary tumors and their metastatic outgrowths.
EMT involves the expression of one or several classical EMT-
TFs, and since the expression of these TFs often accompanies
features of stemness, the retention of this latter state has been
linked to EMT. ZEB1 has been specifically detected in poorly
differentiated pancreatic carcinomas, and it is thought to repress
the expression of stemness-inhibiting miRNAs to maintain a
stem-like phenotype (Wellner et al., 2009). CSC expansion is
also thought to occur after Snail1 mediates a shift from asym-
metric (one stem cell, one differentiating cell) to symmetric
(two stem cells) cell division, implying a role for EMT in increasing
the stem cell pool within the tumor (Hwang et al., 2014). Similarly,
other inducers of EMT, such as the homeobox protein Six1 or
ladybird homeobox-1 protein (LBX1), also induce stemness
and mammary spheroidogenesis when ectopically expressed
in the mouse mammary gland, presumably through their effects
on one of their transcriptional targets (ZEB1, ZEB2, SNAIL1, and
TGFb2) (McCoy et al., 2009; Yu et al., 2009). Nonetheless, it is
noteworthy that the EMT program implicated in normal stem
cells and CSCs may differ, as recently shown in the murine mam-
mary gland, in which they involve Snail2 and Snail1, respectively
(Ye et al., 2015).
Much of the plasticity associated with CSCs arises from the
local niche, both at the primary and secondary tumor sites (Nieto,
2013), and the presumed CSC reservoir is thought to be respon-
sible for tumor relapse and poor clinical outcome. TGFb induces
a program in stromal cells that correlates with poor prognosis
in colorectal cancer patients, in which cancer-associated fibro-
blasts (CAFs) have been linked with an increase in tumor-initi-
ating cells (Calon et al., 2015). Similarly, in hepatocellular carci-
noma that usually develops in the context of fibrosis, CAFs
with characteristics of myofibroblasts promote the appearance
of liver-tumor-initiating cells (Lau et al., 2016). Ongoing molecu-
lar rearrangements were recently proposed to drive adaptive
plasticity in highly metastatic neuroblastoma cells, cells that
strongly express EMT genes and that display a stem-like pheno-
type, with high SOX2 and NANOG expression (Pandian et al.,
2015). Collectively, these studies highlight the regulatory effects
of the microenvironment on cell fate.
Uncoupling EMT and Stemness
Despite the evidence linking stemness and the EMT, recent
studies consider that these two aspects of CSC development
may occur in parallel rather than through the activation of the
same pathway. It is now widely accepted that, although EMT is
instigated in CSCs to drive their dissemination, they undergo
MET on reaching a desirable metastatic niche to seed a new
tumor, presumably by suppressing driver EMT-TFs (Nieto,
2013). The MET at the new metastatic site is surely influenced
by CAFs and a specific type of metastasis-associated macro-
phages (MAMs) (Qian et al., 2015), as well as mesenchymal
stem cells, immunological mediators, and other factors involved
in hypoxia and angiogenesis (Plaks et al., 2015). As such, recent
data indicate that MAMs promote the conversion of hepatic stel-
late cells into myofibroblasts, resulting in a fibrotic microenviron-
ment that sustains metastatic tumor growth in the liver (Nielsen
et al., 2016). Mesenchymal DTCs activate CAFs, which in turn,
promote the transition of DTCs to a more epithelial and prolifer-
ative phenotype that favors metastatic colonization (Del Pozo
Martin et al., 2015).
Parallels can again be seen between the induction of tumor-
initiating capacities in cancer cells and the acquisition of plurip-
otency during fibroblast reprogramming to induced pluripotent
stem cells. The latter also requires a MET (Li et al., 2010b; Sama-
varchi-Tehrani et al., 2010), compatible with the fact that embry-
onic stem cells are epithelial-like. Recent data indicate that the
serial delivery of four transcription factors in a specific order
Oct-4 with Klf4, followed by c-Myc, followed by Sox2 (Liu
et al., 2013b)induces a higher rate of pluripotency. Oct4 first
promotes fibroblasts with an even more mesenchymal-like
phenotype through the upregulation of Snail (Oct4), such that
these cells are then more responsive to Sox2, which is needed
to repress Snail and induce MET in embryos and mouse fibro-
blasts (Acloque et al., 2011; Li et al., 2010b). This delay in MET
is thought to provide time for epigenetic reorganization before
pluripotency is induced (Gaeta et al., 2013). Interestingly, cells
engaged in intermediate reprogramming closely resemble
those found during mesendoderm formation, which is driven
by a MET-like process in mesenchymal mesodermal cells in
the primitive steak (Takahashi et al., 2014). Again this highlights
the similarities with embryonic development and the importance
of intermediate phenotypes in the induction of EMT. A prototyp-
ical example of this is the loss of an epigenetic mark in tropho-
blast epithelial cells that allows them to undergo a partial EMT
while maintaining their self-renewal capacity and multipotency
(Abell et al., 2011).
Further evidence for uncoupling EMT and stemness derives
from studies of the homeobox transcription factor, PRRX1, a
potent EMT inducer in both embryos and cancer cells. In human
breast cancer BT-549 cells, downregulation of PRRX1 is linked
with MET and increased proliferation, but also with a gain in
mammosphere formation and the emergence of a predominant
CD44high cell population (Ocana et al., 2012), all favoring
Cell 166, June 30, 2016 33
metastatic colonization. This again suggests that EMT is not
necessarily associated with stemness. Interestingly, CTGF and
the inhibitor of differentiation (Id) both induce MET and the
expression of pluripotency genes (Chang et al., 2013; Stankic
et al., 2013), which suggest that the morphological phenotype
and stemness can be regulated independently. Indeed, TGFb
is essential for the initial EMT, after which the EGFR-Ras
pathway independently drives stem cell properties (Voon et al.,
2013). This is also consistent with Snail- or Twist-induced
expression of miR-424, which promotes mesenchymal traits
while inhibiting tumor initiation (Drasin et al., 2015). In a more
extreme case, the mutual exclusivity of EMT and stem cell-like
traits is evident in prostate and bladder cancer cell lines, in which
constitutive Snail1 activation suppresses stemness (Celia`-Ter-
rassa et al., 2012). Furthermore, in the skin, low Twist levels
induce tumor initiation and confer stem cell properties while
higher levels are required for the induction of EMT (Beck et al.,
2015). Consistently, Twist-induced tumor-initiating capacity is
only manifested after Twist downregulation at the metastatic
site (Schmidt et al., 2015), and decreasing Snail1, Twist1, or
Prrx1 expression is a requisite for EMT attenuation to allow
CSCs to populate a new metastatic site (Ocana et al., 2012;
Tran et al., 2014; Tsai et al., 2012).
Can Partial EMT States Explain the Stemness-EMT-MET
Link?
The aforementioned data show that EMT and stemness are not
necessarily linked and that the acquisition of both traits can be
uncoupled. However, they also suggest that an intermediate
EMT state may make cells more prone to exhibiting CSC-like
properties. Transient Twist1 activation in mammary epithelial
cells induces their long-term invasive potential, as well as their
capacity to colonize distant organs and tendency to express
traits typical of both epithelial and mesenchymal cells. By
contrast, constitutive Twist1 activation induces a migratory but
not a metastatic phenotype, as cells do not demonstrate tu-
mor-initiating capacities (Schmidt et al., 2015). This is consistent
with the finding that a complete EMT is not required for Twist1-
induced invasion (Shamir et al., 2014) and that plasticity at the
metastatic site does not imply reversion to the fully differentiated
epithelial phenotype (Nieto, 2013). In summary, stemness seems
to be better manifested at intermediate epitheloid states, consis-
tent with those observed in cultured mammospheres (Mani et al.,
2008; Ocana et al., 2012) and in embryonic stem cells.
The intermediate EMT state associated with the stem cell
properties of trophoblast cells (Abell et al., 2011) is similar to
that observed in claudin-low breast cancer cells (Jordan et al.,
2011; Prat et al., 2010). This is also the basis of the proposed ex-
istence of migratory CSCs at the invasive front of tumors (Bra- and Sleeman, 2006), a feature that provides a unique way to tap
bletz et al., 2005). Indeed, transitions between EMT and MET are
likely to be controlled by many factors that sway the balance
from one extreme to the other. Thus, a tissue could display
more mesenchymal or epithelial characteristics at any given
time, yet still with evidence of the other. Indeed, pancreatic can-
cer cells are shown to have stem-like features and an intermedi-
ate EMT phenotype in vivo, with low levels of E-cadherin and
concomitant mesenchymal features (Rhim et al., 2012). Along
similar lines, the upregulation of miR-424 during Twist1- or
Snai1-induced EMT in breast cancer cells can induce mesen-
34 Cell 166, June 30, 2016
chymal genes without affecting the expression of the epithelial
genes (Drasin et al., 2015).
Hybrid, partial transitions in cells lead to an expression of
both epithelial and mesenchymal traits, which can potentially
endow cells with migratory potential while retaining some degree
of cell-cell adhesion, promoting a more efficient coordinated
migration, as observed during embryonic development (Theve-
neau and Mayor, 2011). Stemness is regulated by a LIN28/
let-7 inhibitory loop (Yang et al., 2010b), with a balanced, inter-
mediate level of the two helping to maintain an intermediate level
of Oct4 for stemness (Niwa et al., 2000). Building on this, a
stemness window that regulates the interplay between
LIN28 and Let-7 has been proposed to control stemness and
EMT (Jolly et al., 2015). Through the use of theoretical framework
modeling, it was shown that strong inhibition of LIN28 by miR-
200 pushes the stemness window toward the mesenchymal
end of the EMT axis, whereas strong inhibition of ZEB by Let-7
forces it in the other direction. From this, it was suggested that
stemness can be gained by cells with different phenotypes
based on the actions of Let-7 and Lin28 but also in response
to changes in other signals like Snail1 and, presumably, Twist
or other prominent inducers of EMT.
A sub-population of metastasis stem cells was recently
described among the CTC population (Baccelli et al., 2013),
although it remains unclear whether CTCs harbor a small fraction
of CSCs or if CTCs derive from cells with the potential to become
CSCs, themselves a separate entity. Resistance to treatment
may be acquired at metastatic sites in micrometastatic stages.
Therefore, it will be necessary to determine whether stemness
is a property of subsets of CTCs or whether EMT is solely a
mechanism critical for dissemination, with stemness re-acquired
at the site of arrest in the parenchyma of the target organ through
MET (Beck et al., 2015; Brabletz, 2012; Jung and Yang, 2015;
Polyak and Weinberg, 2009). Stemness, or the attainment of
stem cell properties, is no longer considered a fixed trait,
with such cells displaying a high degree of plasticity (Jolly
et al., 2015). Stemness is well defined in embryonic development
and it is a hallmark of cancer within a particular window during
disease progression. Like other hallmarks of cancer, stemness
can be lost or acquired, and it may be affected by the expression
of certain phenotypic traits as cancer cells and tumors progress
toward the metastatic disease (Antoniou et al., 2013).
The Spectrum of EMT Plasticity in Cancer: Therapeutic
Challenges
Re-differentiation represents an attractive therapeutic option to
reverse EMT (Beug, 2009; Huang et al., 2012; Nieto, 2013; Thiery
into current mainstream therapies (Chua et al., 2011). However,
there are several concerns that must first be considered. Given
the existence of EMT gradients, we have yet to determine how
far the cells should be reversed. Furthermore, it remains unclear
what specific EMT score would be effective to abolish metas-
tasis and/or enhance drug sensitivity. Evidence indicates that a
full EMT reversal might not be optimal for metastatic coloniza-
tion, and thus, the effective shift is likely to be context dependent
and based on the different patterns of EMT in distinct cancer
types. Indeed, the re-expression of E-cadherin in fibroblasts
does not reverse them to the epithelial state (Navarro et al.,
1993), and while restoring E-cadherin to ovarian cancer cells in
culture abolishes their invasive properties, their mesenchymal
traits remain unaffected (Huang et al., 2013).
The drug discovery platform for EMT reversal is limited,
although it is feasible to conceive drug pipelines that preferen-
tially induce cytotoxicity in cells that underwent EMT. Lead com-
pounds to target CSCs in breast cancers have been identified
in screening programs based on EMT models, such as human
mammary epithelial cells bearing shRNA against E-cadherin
(Carmody et al., 2012; Gupta et al., 2009b). There are also pipe-
lines for anti-cancer treatments that target specific components
of signaling pathways, including HGFR, insulin-like growth fac-
tor-1 receptor (IGF-1R), EGFR, PDGFR, TGFbR, phosphatidyli-
nositol 3-kinase (PI3K)/Akt, and ERK/MAPK (Lamouille et al.,
2014). While these treatments could be considered as anti-
EMT drugs, these pipelines were not designed with a specific
anti-EMT concept in mind, and therefore, the anti-proliferative
and EMT-specific effects of these inhibitors might be difficult
to assess independently. Few platforms have been developed
that screen for non-cytotoxic agents that reverse EMT. A
screening model based on a growth-factor-induced scatter
assay (including HGF, IGF-1, and EGF) in NBT-2 rat bladder car-
cinoma cells has been adapted for this purpose (Chua et al.,
2012). Its use highlighted compounds that target activin A recep-
tor type II-like kinase (ALK5)/TGFbR1, MAPK, Src, and PI3K,
none of which exhibited significant effects against proliferation.
However, this model requires additional validation in a second-
ary screen on human carcinomas. Recent data indicate that
activation of the PKA pathway induces epigenetic changes
that can modulate EMT together with the loss of tumor-initiating
capacities in a xenograft breast cancer model (Pattabiraman
et al., 2016).
TGFb inhibitors are the most intensively investigated anti-
EMT compounds, and recent Phase I studies tested the use of
LY2157299 as a TGFb inhibitor in glioblastoma and hepatocellu-
lar carcinoma (Giannelli et al., 2014; Rodon et al., 2015). How-
ever, the effect of LY2157299 on EMT reversal warrants further
study. It is worth noting here that TGFb can also act as a tumor
suppressor through the activation of a particular type of lethal
EMT in pancreatic cancer cells (David et al., 2016). Src kinase in-
hibitors also clearly reverse EMT (Huang et al., 2013; Vultur et al.,
2008), yet their clinical effects as a monotherapy or in combina-
tion with other agents have been disappointing (Kim et al., 2009;
Puls et al., 2011). An inhibitor of the closely related focal adhe-
sion kinase (FAK), PF-00562271, is also being tested in a
Phase I dose-escalation trial against solid tumors with promising
results (Infante et al., 2012). These Src and FAK inhibitors must
undergo lethality screens to determine the appropriate combi-
nations of the compounds to induce EMT reversal. These drug
screening platforms suffer from the same lack of microenviron-
mental modeling as cell culture models. However, microfluidic
co-culture systems have recently been developed to mimic
the tumor microenvironment and induce the formation of
3-dimensional tumor spheroids in order to screen for EMT-
blocking agents (Aref et al., 2013). This type of model more
accurately assesses the efficacy of EMT reversal agents to
combat the appearance of sprouting endothelial cells and/or
immune cells. Finding a way to eradicate these epithelial cells
would identify a mechanism through which the lethality of the
cells can be enhanced following EMT reversal. One example
would be the use of epigenetic modifiers, such as histone de-
acetylase (HDAC) inhibitors, to reverse ZEB1-regulated EMT
and sensitize pancreatic cancers to gemcitabine (Meidhof
et al., 2015). This strategy could be applied in the front-line or
in chemoresistance settings.
Finally, anti-EMT therapeutics should be carefully defined and
positioned. Should we reverse or kill the cells that underwent
EMT? Are we comfortable with reversing EMT and maintaining
a stable disease status with these cells regaining an epithelial-
like phenotype since they are less migratory and invasive? An
important note of caution here comes from the evidence that
reversal is also required for metastatic colonization in vivo (Alder-
ton, 2013; Brabletz, 2012; Ocana et al., 2012; Tsai et al., 2012;
Beerling et al., 2016)). Therefore, promoting EMT reversal in-
cludes the potential risk of enhancing the establishment of sec-
ondary metastasis by DTCs (Nieto, 2013). Thus, it will be imper-
ative to either define a proper therapeutic window in which to
administer an EMT-reversing agent, or alternatively, the goal
should be to eradicate the cells that underwent EMT. If effective,
specifically eliminating those cells appears to be the optimal
strategy. Even in the absence of metastatic disease, most locally
advanced patients still relapse, and it is known that recurrence is
accompanied by EMT, with Snail1 proving sufficient to promote
mammary tumor recurrence in vivo (Moody et al., 2005). Interest-
ingly, specifically targeting cells that underwent EMT might also
target dormant cells in distant organs, as they are likely to be in a
mesenchymal state. As such, NR2F1 has recently been consid-
ered a critical node in the induction of dormancy in DTCs (Sosa
et al., 2015). Interestingly, NR2F1 is known to lie at the core of the
EMT regulatory chromatin landscape in the neural crest (Rada-
Iglesias et al., 2012).
EMT Confers Resistance to Chemotherapy and
Immunotherapy
EMT confers resistance to cell death induced through various
means both in embryos and in cancer cells (Vega et al., 2004;
Thiery et al., 2009), including chemotherapy (Singh and Settle-
man, 2010). Even in studies that find a limited contribution of
cells that have undergone EMT to established metastases, the
role of EMT in conferring chemoresistance is clear (Fischer
et al., 2015; Zheng et al., 2015). Therapeutic strategies should
therefore encompass an arm that targets network rewiring
when resistance emerges. For instance, a rewiring event occurs
in EGFR-mutated non-small-cell lung carcinoma (NSCLC) that
switches EGFR to Axl receptor tyrosine kinase in association
with a mesenchymal phenotype (Byers et al., 2013). One can
therefore propose targeting these erlotinib-resistant mesen-
chymal NSCLC cells with potent inhibitors of Axl (Sheridan,
2013), although it might also be envisaged that resistance to
Axl will be acquired when network rewiring repeats. Neverthe-
less, there is increased interest in targeting Axl signaling (Gjer-
drum et al., 2010) as a first-in-line class of therapeutic agent
for EMT (clinicaltrials.gov; NCT02488408).
Immunotherapy for cancer treatment has generated much in-
terest in recent years, with notable successes against several
Cell 166, June 30, 2016 35
tumors, including melanoma, NSCLCs, and renal carcinoma.
These approaches are based on the blockade of immune inhib-
itory checkpoints, such as the use of monoclonal antibodies to
programmed death-1 (PD1) or to cytotoxic T lymphocyte-asso-
ciated antigen 4 (CTL4), two inhibitory T cell receptors (Okazaki
et al., 2013). Further improvement in these therapeutic interven-
tions would permit longer survival than that achieved using tar-
geted therapeutics. Numerous somatic mutationssome of
which are considered to be trunk and branch drivershave
been described in many tumors. However, new, dominating mu-
tations are often induced by conventional and targeted thera-
peutics, and they have the potential to make cells refractory to
these treatments (McGranahan and Swanton, 2015). In principle,
although such rapid drift in mutation profiles may not affect CTL
cancer cell lysis, EMT of the target cancer cell may potentially
abrogate CTL lysis.
Melanoma cells that undergo Snail-mediated EMT are much
more metastatic than their parental cells, which was attributed
to the emergence of T regulatory (Treg) CD4 cells expressing
Foxp3, a known inducer of immunosuppression in Treg cells
(Kudo-Saito et al., 2009). The emergence of these Treg cells
was in part driven by the TGFb and thrombospondin secreted
by these Snail1-expressing melanoma cells. Furthermore, a
Snail small interfering RNA (siRNA) injected in vivo reduced the
immunosuppressive and metastatic potential of melanoma cells
(Kudo-Saito et al., 2009).
Plasticity of carcinoma cell phenotypes can be achieved by
conventional and targeted therapies and through the tumor
microenvironment (Holzel et al., 2013). Thus, as well as abro-
gating immune suppression in CTLs, one might consider modi-
fying the EMT phenotype of carcinoma cells. Indeed, CTL lysis
of carcinoma cells was considerably reduced when Snail-medi-
ated EMT of MCF7 cells occurred in response to chronic expo-
sure to TNFa due to the induction of autophagy (Akalay et al.,
2013). EMT of the target cells affects the maturation of the
immune synapse, although defining the mechanism that drives
this plasticity in the cell cortex requires further study. It must
also be ascertained what causes the defects in the organization
and positioning of T cells, and of the adhesive receptors ar-
ranged as concentric rings in the immune synapse (Chouaib
et al., 2014). Intriguingly, tumors that respond best to CTL-A4,
and more recently to PD1 and PD1L antibodies, exhibit a higher
EMT score (Tan et al., 2014). These tumors include melanomas
and renal, bladder, and NSCL carcinomas (Topalian et al.,
2012; Motzer et al., 2015; Rizvi et al., 2015). Thus, these anti-
bodies could specifically restore a functional immune synapse
in carcinomas that exhibit a mesenchymal- rather than an epithe-
lial-like phenotype (Engl et al., 2015). This proposal is supported
by recent data indicating that an increase in target cell tension
potentiates killing by CTLs (Basu et al., 2016).
In addition to immunotherapy, a vaccine was recently devel-
oped against a mesenchymal-associated T-box TF, brachyury
(Hamilton et al., 2013), which is currently undergoing clinical tri-
als for the treatment of solid tumors. In general, these fast-devel-
oping fields in immunotherapy and cancer vaccine development
will most likely provide additional therapeutic options and in-
sights into how to target and eradicate the population of cells
that have undergone EMT in cancer.
36 Cell 166, June 30, 2016
Challenges, Future Directions, and Perspectives
Originally described as a major event in embryonic morphogen-
esis, EMT is successfully recapitulated in various normal and
abnormal processes, a phenomenon that has become well
accepted since it was first proposed in the early 2000s. Yet,
despite extensive forays into the role of EMT in disease progres-
sion, we are still far from reaching a consensus as to the true
role EMT plays in these processes. While is seems clear that
reversing EMT is beneficial for the treatment of fibrosis, the
inherent complexity of cancer suggests that a better strategy
may be to eradicate the cells that underwent EMT in the carci-
noma. However, this still requires the development of better
animal models together with technological advances to better
visualize the EMT in vivo at the organ, tissue, cellular, and sub-
cellular levels including advanced genomics in FACS-enriched
cell populations. These approaches will directly test some of
the concepts discussed here and help comprehend the dy-
namics of EMT to better design anticancer therapies.
An emerging challenge will be to decipher inter- and intratu-
moral heterogeneity, including the full spectrum of intermediate
EMT states and how cells make the transitions between these
states. Targeted therapeutics face the formidable challenge
of rapid tumor evolution, as revealed by the genomic land-
scape of distinct biopsies in primary and metastatic tumors
(McGranahan and Swanton, 2015). It is also important to eval-
uate to what extent stochastic events could generate cells with
a stem-cell-like phenotype and how the cell of origin influences
the susceptibility to undergo EMT. Another important issue is
to better understand the mechanisms for lymphatic dissemina-
tion in different cancer types. Although the EMT does not seem
to be required for lymph node colonization (Giampieri et al.,
2009), its relative contribution versus collective cell migration
deserves further investigation.
Bioinformatics algorithms must be developed to go far beyond
gene ontology, principal component analyses, and the currently
available networks. Most importantly, the predictions from math-
ematical models and hypothetical gene regulatory networks
need to be tested in appropriate animal models. Furthermore,
powerful bioinformatics analysis can also help in the new ave-
nues that link EMT with metabolic changes (Choudhary et al.,
2016; Jiang et al., 2015; Mathow et al., 2015; Shaul et al., 2014).
Intravasation has been the least studied process in the meta-
static cascade, and in light of recent data that suggests that EMT
may be more limited than previously anticipated (Fischer et al.,
2015; Zheng et al., 2015), attention should be paid to the possi-
bility of cancer epithelial cells being able to efficiently intrava-
sate. Do just a few mesenchymal cells help epithelial cancer cells
intravasate? Is the leakiness of the tumor vessels sufficient to
allow intravasation of epithelial cell clusters? As intravasation is
clearly a crucial step in dissemination, further attention should
be now paid to this process, particularly probing the recent pro-
posal that TAMs and vascular mimicry acquired by cancinoma
cells help them to intravasate (Harney et al., 2015; Wagenblast
et al., 2015).
It will also be relevant to define at what point in the lifespan of a
CTC the cell undergoes a MET-like process: before it reaches the
metastatic site (i.e., in the bloodstream) or after the metastasis
has started to grow at a new site. Answering this question will
surely be aided by identifying better markers for CTCs, particu-
larly as EpCAM fails to detect all CTC states. It is also important
to ask whether CSCs travel with CTCs in collective migration, the
extent to which CSCs convert into non-CSCs (and vice versa)
(Chaffer et al., 2011; Schwitalla et al., 2013), and how is such
plasticity controlled.
Cancer stroma crosstalk appears to be context specific, and
thus, we must better understand the nature of the stroma that
respectively promotes or represses EMT in the primary tumor
or in the metastatic niche. In relation to this, the description of
a pre-metastatic niche has been an important development (Ka-
plan et al., 2006), as has the role of membrane vesicles derived
from tumor cells, called exosomes, which can induce vascular
leakiness and bone marrow progenitor mobilization to educate
the niche (Peinado et al., 2012). Exosomes derived from pancre-
atic ductal carcinoma can initiate the pre-metastatic niche in the
liver (Costa-Silva et al., 2015). The target cells are the Kupffer
cells, which instruct stellate cells to transdifferentiate into myofi-
broblasts and generate a fibrotic microenvironment that favors
the formation of the metastatic niche. This is reminiscent of the
situation in renal fibrosis, where damaged epithelial cells secrete
exosomes that initiate the differentiation of fibroblasts into
myofibroblasts (Borges et al., 2013). Importantly, the homing
specificity of exosomes directed by the expression of different
integrins defines the target tissue where cancer cells will prefer-
entially form metastatic outgrowths (Hoshino et al., 2015). Thus,
a deeper analysis of the metastatic niche and its interaction with
exosomes, DTCs, and dormant cancer cells is now warranted.
Most intriguing is how we can target the EMT program for ther-
apeutic gain. Specific deletion of the cells that have undergone
EMT would be optimal to prevent the colonization of distant sites
by newly generated DTCs or long-standing dormant cells. This
implies specifically rendering those cells sensitive to cytotoxic
drugs, which is a challenge if we consider that they are resistant
to multiple death-inducing signals and also to chemo- and
immunotherapy (Fischer et al., 2015; Kudo-Saito et al., 2009;
Voon et al., 2013; Zheng et al., 2015). Many of these challenges
will require innovative approaches.
We are at the dawn of a new era of personalized medicine and
the recent advances in pharmacogenomics will provide clear-cut
opportunities to tailor individualized cancer treatment strategies.
With better treatments for cancer should come additional in-
sights into how cancer progresses, and knowledge as to which
are the most relevant drivers, pathways, and modes of dissem-
ination. These advances may help clarify the extent to which
EMT is involved in the various disease states and point to
avenues through which our current understanding of the EMT
pathway and transitional events can be exploited to target tu-
mors and/or make them more susceptible to treatment regimes.
ACKNOWLEDGMENTS
We sincerely apologize to colleagues whose work has been omitted from this
review owing to space limitations. We thank Stuart Ingham from the Instituto
de Neurociencias for his help with the figures. This work has been supported
by grants from the Spanish Ministry of Economy and Competitiveness
(MINECO-BFU2014-53128-R), the Generalitat Valenciana (Prometeo II/2013/
002 and ISIC 2012/010), and the European Research Council (ERC
AdG322694) to M.A.N. and from Department of Biochemistry and Cancer Sci-
ence Institute National University of Singapore and Institute of Molecular Cell
Biology A*STAR Singapore to J.P.T. The Instituto de Neurociencias is a Centre
of Excellence Severo Ochoa. This review is dedicated to the late Professor
Gerald Maurice Edelman who was very influential on the research carried
out by J.P.T. and to Mr. Angel Nieto who passed away recently and always
was a role model for M.A.N.
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