DEVELOPMENTAL DYNAMICS 248:10–20, 2019

DOI: 10.1002/dvdy.24678

REVIEW

Epithelial-mesenchymal Transition and Cancer

Stem Cells: At the Crossroads of Differentiation
and Dedifferentiation
Hanmin Wang and Juli J. Unternaehrer*

Division of Biochemistry, Department of Basic Sciences, Loma Linda University, Loma Linda, California

In this review, we explore the connections between epithelial-mesenchymal transition (EMT) and differentiation status. EMTs
in development have been described as differentiation events, while in most cases EMTs in cancer have been depicted as
dedifferentiation events. We will briefly summarize both embryo development and cancer progression with regard to the
involvement of EMT and cell differentiation status. We further present the studies that provide evidence that EMT results in
both differentiation and dedifferentiation. Finally, we present our resolution to this dilemma by suggesting that EMT brings
about dedifferentiation that enables subsequent differentiation. In normal development, EMT events may cause a partial rever-
sal of differentiation to overcome differentiation barriers. When EMT is aberrantly activated in cancer, cells gain attributes of
stem cells that contribute to self-renewal capabilities and are able to differentiate to all cell types represented in the tumor.
The resulting cancer stem cells attain hallmarks of cancer, including replicative immortality, resistance to cell death, and inva-
siveness. Developmental Dynamics 248:10–20, 2019. © 2018 Wiley Periodicals, Inc.
DEVELOPMENTAL DYNAMICS

Key words: development; reprogramming; miRNA regulation; epithelial-mesenchymal transition; differentiation

Submitted 29 January 2018; First Decision 29 May 2018; Accepted 27 September 2018; Published online 22 November 2018

Introduction
Stemness refers to a cell’s ability to self-renew and differentiate.
It is a characteristic of all stem cell categories, including embry-
onic, adult, and cancer stem cells (Batlle and Clevers, 2017). Cells
with the greatest degree of stemness are the least differentiated.
Many tumors adopt an oncofetal gene expression pattern, with
reversion to an embryonic phenotype, including expression of plur-
ipotency genes (Al-Hajj et al., 2003; Schatton et al., 2008). Pluripo-
tency refers to a cell’s ability to differentiate into all embryonic
lineages and is best illustrated by embryonic stem (ESCs) and
embryonal carcinoma cells. An illustration of cell states discussed
in this review is shown in Figure 1. Based on the degree of stem-
ness (developmental potential), cell states are categorized as totipo-
tent, pluripotent, multipotent and unipotent (Fig. 1).
In addition to the involvement of stem cells, embryo develop-
ment and cancer progression share other similarities. One com-
mon feature is a process called epithelial-mesenchymal transition
(EMT) (Hay, 1995; Martin-Belmonte and Perez-Moreno, 2011;
Thiery et al., 2009). Through EMT, epithelial cells lose their cell
polarity and cell-cell adhesion, and gain migratory and invasive
features to become mesenchymal cells. EMT is essential for
embryogenesis, which has typically been described as a gradual
process of differentiation (Bell and Watson, 2009; Lim and Thiery,
2012). The steps involving EMT are thus suggested to be part of
the loss of potency (differentiation potential) associated with
development. However, some data describe cells that have under-
gone EMT as more stem cell–like than their progenitors, implying
that EMT causes dedifferentiation (Thiery, 2002). These changes
are caused by the action of several transcription factors, including
Snai1 (Snail), Snai2 (Slug), Twist, ZEB1, and ZEB2, which are
potent repressors of epithelial gene expression (Batlle et al., 2000;
Bolós et al., 2003; Cano et al., 2000; Comijn et al., 2001; Hajra
et al., 2002; Yang et al., 2004). These factors also have actions
independent of the EMT, thus the process of EMT must be distin-
guished from the expression of transcription factors mediating
EMT (Vega et al., 2004; Vesuna et al., 2008; Zhang et al., 2015).
We compare the complete dedifferentiation that occurs in somatic
cell reprogramming (Takahashi and Yamanaka, 2006) to the par-
tial dedifferentiation that occurs in EMT (Fig. 1). In this review,
we focus on the hierarchical relationships between these states
and examine the role of EMT in state transitions.
In general, EMTs in development have been described as dif-
ferentiation events, while EMT in cancer has been depicted as a
dedifferentiation event. The end result of the EMT-associated
processes might partially cause this dichotomy. In the following
sections, we summarize the apparently contradictory literature

*Correspondence to: Juli Unternaehrer, Assistant Professor, Department of

Basic Sciences, Division of Biochemistry, Loma Linda University School of Article is online at: http://onlinelibrary.wiley.com/doi/10.1002/dvdy.
Medicine, 11085 Campus Street, Mortensen Hall 219, Loma Linda, CA 24678/abstract
92354. E-mail: junternaehrer@llu.edu © 2018 Wiley Periodicals, Inc.

10

pointing to EMT as differentiation vs. dedifferentiation events.
We present a synopsis of the literature pertaining to EMT and
the differentiation state in an attempt to reconcile these bodies
of literature. In the end, we postulate that although the overall
trajectory of developmental processes is toward differentiation,
cells partially and transiently dedifferentiate when they undergo
EMT. This loss of differentiation represents an opportunity for
adjusting gene expression programs and allows new gene
expression required for formation of new tissue types in devel-
opment. Similarly, the transient loss of differentiation seen in
cancer-associated EMT provides a window of opportunity for
acquisition of a stem cell phenotype. Thus, stem cells or differen-
tiated cells in which EMT transcription factors are active may
simultaneously receive signals leading to the loss of key factors
responsible for maintaining differentiation.
EMT in Development
EMT is essential for many developmental processes and plays a
role in embryonic differentiation in many contexts across the
spectrum of metazoans (Thiery and Sleeman, 2006). EMT is
involved in the initiation of placenta formation and the implanta-
DEVELOPMENTAL DYNAMICS
tion of the embryo (Vicovac and Aplin, 1996). Subsequently, dur-
ing gastrulation, cells in the primitive streak (or comparable
structures such as the ventral furrow in Drosophila, blastoderm
margin in fish) undergo EMT to cause the ingression that leads to
germ layer and mesenchyme formation in metazoans (Baum
et al., 2008; Leptin and Grunewald, 1990). Through invagination,
at the primitive streak, the epiblast generates mesendoderm, which
further separates into mesoderm and endoderm through EMT (Lim
and Thiery, 2012). In development, Snail expression is detected as
early as the two-cell stage in mammalian embryos (Bell and Wat-
son, 2009). Snail is expressed in a variety of species: It is
expressed during the formation of the ventral furrow in Drosoph-
ila (Leptin and Grunewald, 1990); it was also found in stroma of
the choroid plexus of both the hindbrain and the forebrain in
chicken embryo development (Marin and Nieto, 2004). An ortho-
log of Snail is critical for primary mesenchyme cell ingression in
sea urchin embryo (Wu and McClay, 2007). Snail mutant mouse
embryos do not survive gastrulation, confirming Snail’s impor-
tance in early development (Carver et al., 2001). A conditional
knockout of Snail in epiblast, however, affects only the mesoderm
migration, not its initial formation (Lomelí et al., 2009; Murray
and Gridley, 2006). Thus, multiple functions of EMT can be teased
out, and differentiation can occur separately from migration. The
biological importance of Snail is illustrated by the conservation of
its function in a wide spectrum of species.
In the following sections, we will present EMT events involved
in embryo development that are linked to either cell differentia-
tion or cell dedifferentiation.
Evidence that EMT Results in Differentiation
Several events in natural development suggest that EMT results in
differentiation (Fig. 2A). For instance, when cells invaginate from
the primitive streak to form the mesoderm through EMT, they dif-
ferentiate into lineages including blood (Eichmann et al., 2005;
Fehling et al., 2003), skeletal muscle (Pu et al., 2015), and skin
(Nitzan and Kalcheim, 2013). In the general process of embryo-
genesis, after an EMT event, differentiation of the newly produced
mesenchyme follows. The highest diversity in cell lineage
EMT IN DIFFERENTIATION AND DEDIFFERENTIATION 11
differentiation occurs in the mesenchyme derived from the chro-
nologically earliest EMT (Pérez-Pomares and Muñoz-Chápuli,
2002). However, this developmental potential progressively
decreases in later EMTs. In addition, endocardial cushion mesen-
chyme is formed from endocardium cells through another round
of EMT (Carmona et al., 2000); cell differentiation happens during
this process (Romano and Runyan, 1999).
EMT is essential for neural crest formation. In this process, cells
undergo EMT and delaminate from the neuroepithelium, enabling
them to migrate and form the neural crest progeny, including
neurons, endocrine cells, cartilage, bone, dermis, connective tis-
sue, and adipose tissues in the head and neck (Christ et al., 1983;
Dupin et al., 2007). Because of their remarkable differentiation
potential, the neural crest has been called the “fourth germ layer”
(Hall, 2000). In addition, neural crest delamination represents one
of the most distinct developmental mechanisms that involves
EMT. Neural crest cells originate in the neural fold or the neural
tube, then migrate and delaminate as mesenchymal-like cells to
target locations, where they differentiate into various cell
types (Gammill and Bronner-Fraser, 2003; Nikitina et al., 2008;
Steventon et al., 2005; Weston and Thiery, 2015).
Further EMTs happen throughout development, for example,
in the formation of pancreas, kidney and liver; in the somites to
form sclerotome and myotome; and in cardiac development
(Costello et al., 2011; Nakajima et al., 2000). To better under-
stand EMT in development, it is also important to consider
mesenchymal-epithelial transition (MET), the reverse process of
EMT (Lim and Thiery, 2012). Mesenchymal cells originating from
the primitive streak take part in the formation of epithelial
mesodermal organs like somites through MET. Waves of EMT
and MET occur repeatedly during organogenesis (Lim and Thi-
ery, 2012). During this process, cells disseminate to various parts
of the embryo and differentiate into many types of cells.
Heart formation is an illustrative example of organogenesis
that involves multiple EMTs. Cardiac-specified cells first undergo
EMT to migrate rostrally, and a subsequent MET leads to the for-
mation of two cardiogenic territories, followed by the heart pri-
mordium formation. Then, a second cycle of EMT/MET creates
the endothelial cell lining of the heart. A third cycle creates the
endocardial cushion and its derivatives. A cluster of mesothelial-
derived cells undergoes another MET to form epicardium. During
the following, fourth cycle, epicardial cells delaminate and dif-
ferentiate into epicardial-derived cells—mesenchymal-like cells
that further populate the subepicardium—and are responsible for
the formation of endothelial cells, coronary smooth muscle, and
cardiac fibroblasts (Chua et al., 2011). It takes four cycles of
EMT for progenitor cells to form cardiac fibroblasts; after each
cycle, cells differentiate into a more specialized cell type.
EMT-related gene expression changes may also provide input
to the differentiation state. Cells that undergo EMT change their
adhesion-molecule gene expression, down-regulating epithelial
factors, such as E-cadherin, occludin, and Traffic Jam 1 (TJ1),
and up-regulating those associated with the mesenchymal state,
such as N-cadherin (Cano et al., 2000; Hazan et al., 1997; Islam
et al., 1996; Kim et al., 2000). Snail promotes the cadherin
switch from E-cadherin to N-cadherin expression (Oda et al.,
1998). The expression of N-cadherin plays roles in differentia-
tion: For example, N-cadherin expression controls myogenic dif-
ferentiation (Charrasse et al., 2002). Recently, Schafer
et al. suggested that, in addition to the surface adhesion changes
caused by the cadherin switch, Snail may serve to facilitate
 12 WANG AND UNTERNAEHRER

Fig. 1. Epithelial-mesenchymal transitions in Waddington’s epigenetic landscape. Dashed arrows indicate state changes. Solid blue lines indicate
added differentiation potential of cells after EMT. EMT is proposed to be associated with partial dedifferentiation, resulting in cells with increased
stemness. In development, resulting new gene expression allows cells to differentiate into new cell types. In cancer, dedifferentiation endows cells
with self-renewal abilities and ability to regenerate all tumor cell types. Terminally differentiated cells (green to yellow) or multipotent progenitors (red
to pink) can undergo EMT and be partially reprogrammed. Classical reprogramming to pluripotency (e.g., by Yamanaka factors) is indicated by green
to blue, partial reprogramming by green to red or yellow, and direct reprogramming by green to green lines. Adapted from Waddington (Waddington,
1957) and Hochedlinger and Plath (Hochedlinger and Plath, 2009), with permission.
DEVELOPMENTAL DYNAMICS

downstream events such as Wnt signaling (Schäfer et al., 2014).
The Wnt signaling pathway is well known to participate in cell
proliferation and cell differentiation (Reya and Clevers, 2005).
These gene expression changes hence indicate cell differentiation
after EMT.
Evidence from pluripotent cell biology also supports the con-
cept that EMT leads to differentiation. In ESCs, which display
many but not all characteristics of bona fide epithelium, sponta-
neously differentiating cells have been described to undergo
EMT at the periphery of colonies (Ullmann et al., 2007). This is
thought to be analogous to gastrulation (Kim et al., 2014). When
human ESCs differentiate, they undergo EMT (Li et al., 2017).
Galvagni et al. (2015) used ESCs expressing tamoxifen-inducible
Snail1 to investigate the role of Snail1 expression in early ESC
differentiation. Eight hours after induction, they analyzed gene
expression and found 17 self-renewal-related genes were down-
regulated, while the up-regulated genes are normally expressed
in later-stage, more differentiated stem cells. They conclude that
Snail1 expression induces ESC exit from pluripotency. In this
case, the EMT was associated with differentiation.
All the developmental events mentioned previously link EMT
to differentiation.
Evidence that EMT Results in Dedifferentiation
Unlike the evidence presented earlier, there are studies that define
EMT as a dedifferentiation event. In embryo development, another
member of the Snail family, Slug, acts in concert with Sox9 to
induce differentiated luminal cells of the mammary gland to
acquire stem cell characteristics (Guo et al., 2012). Twist acts to
maintain the immature or dedifferentiated phenotype, and its loss
allows myoblast, chondrocyte, and osteoblast differentiation
(Alborzi et al., 1996; Dong et al., 2007; Hebrok et al., 1994). Over-
expression of Snail or Twist in breast epithelial cells results in
cells with increased differentiation potential, similar to mesenchy-
mal stem cells (MSCs) (Battula et al., 2010). Snail overexpression
in MSCs blocks differentiation to bone and adipocyte fate (Batlle
et al., 2013); similarly, Twist overexpression in MSCs results in
maintenance of an immature phenotype and prevents differentia-
tion to bone and cartilage, but allows differentiation to adipose
tissue (Isenmann et al., 2009). These data implicate Snail and
Twist in maintaining the dedifferentiated state.
Several observations from the reprogramming field also provide
evidence that dedifferentiation results when EMT factors are
expressed. Somatic cells can be dedifferentiated, or reprogrammed,
to pluripotency by the expression of the four “Yamanaka” factors
(Park et al., 2008; Takahashi et al., 2007; Takahashi and Yamanaka,
2006; Yu et al., 2007). Reprogramming is a multistep process
involving both EMT and MET that requires many days to accom-
plish (Samavarchi-Tehrani et al., 2010). Snail and other mesenchy-
mal gene expression increase early in fibroblast and keratinocyte
reprogramming before waning as the reprogrammed cells take on
an epithelial fate (Samavarchi-Tehrani et al., 2010). An early induc-
tion of EMT, either by sequential application of reprogramming fac-
tors, transient Transforming Growth Factor-beta (TGF-β) treatment,
or expression of Snail or Slug, improves reprogramming efficiency
(Liu et al., 2013). Inhibitors of TGFb signaling, and thus of EMT,
antagonize reprogramming when administered prior to the repro-
gramming factors; later, they enhance the process as expected, since
they promote MET (Maherali and Hochedlinger, 2009). Consistent
with these findings, reprogramming efficiency is decreased by
inhibiting, and increased by overexpressing, Snail (Unternaehrer
et al., 2014). Thus, EMT appears to enhance early steps of
dedifferentiation.
An important signaling pathway in pluripotency and early
embryo development is the Wnt pathway, which exerts pleiotro-
pic effects dependent on context. Because of the involvement of
Wnt signaling in both stemness and EMT (Fabregat et al., 2016;
Gonzalez and Medici, 2014; Micalizzi et al., 2010), studies on
this pathway provide insight to how the two processes relate.
Wnt signaling maintains pluripotency and the self-renewal abil-
ity of mouse ESCs (Anton et al., 2007; Bose et al., 2007; Essafi
et al., 2011). A higher percentage of ESCs formed colonies when
plated in the presence of Wnt3a protein, whereas the Wnt

DEVELOPMENTAL DYNAMICS
Fig. 2. Diagram of epithelial-mesenchymal transition effect on
differentiation status. A: Many findings suggest that EMT results in
differentiation. During the process of EMT, cells lose stemness and
undergo differentiation. The yellow end of the spectrum represents the
stem cell fate; the blue represents differentiated cells. B: Other findings
suggest that EMT results in dedifferentiation. Some studies have
concluded the opposite, that during the process of EMT, cells
dedifferentiate and attain stem cell–like features. C: The data are
consistent with a third explanation, that EMT, especially partial EMT,
results in transient dedifferentiation allowing differentiation. The action of
EMT transcription factors may bring about a reprogramming event
resulting in dedifferentiation. The resulting stem-like cells attain self-
renewal capabilities and display increased differentiation potential.
antagonist reduced, and at high concentration completely sup-
pressed, self-renewal (ten Berge et al., 2011). Furthermore, the
effect was rescued by addition of Wnt3a protein. Wnt signals are
hence proved to be essential self-renewal factors for ESCs and
are required to inhibit their differentiation into more advanced
epiblast-derived stem cells (ten Berge et al., 2011). In addition,
Wnt signaling increases the efficiency with which differentiated
cells can be reprogrammed toward pluripotency (Lluis et al.,
2008; Marson et al., 2008).
Wnt signaling is also crucially involved in EMT (ten Berge et al.,
2008; Lindsley et al., 2006). The Wnt signaling pathway induces
EMT in various models including colon- and breast-carcinoma cell
lines and mammary epithelial cells (Kim et al., 2002; Yook et al.,
2006). Furthermore, in breast cancer patients, markers indicating
active Wnt signaling also correlate clinically with poor prognosis
(Logullo et al., 2010; Prasad et al., 2009). Another study showed
Wnt signaling engages tumor cell dedifferentiation while stabilizing
Snail, further inducing EMT (Yook et al., 2006). The fact that Wnt
EMT IN DIFFERENTIATION AND DEDIFFERENTIATION 13
signaling results in both stemness and EMT draws a further con-
nection between these phenomena.
Taken together, these findings point to a role for EMT factors
in the acquisition of pluripotency.
EMT in Cancer
EMT also occurs in cancer cells: Tumor cells recapitulate ontog-
eny, thus inappropriately activating EMT (Thiery, 2002). Transi-
tions between epithelial and mesenchymal states in cancer cells
are linked with their ability to spread (Peinado et al., 2007;
Polyak and Weinberg, 2009). A subpopulation of cancer cells in
primary tumors undergoes EMT, resulting in enhanced migratory
and invasive properties. This is an early step of establishing a
metastasis and forming secondary tumors (Thiery, 2002).
Although developmental EMT is described either as differentia-
tion or dedifferentiation, cancer studies have almost exclusively
depicted EMT as dedifferentiation (Fig. 2B).
EMTs Result in Acquisition of Stemness in Cancer Cells
Rare tumor cells possess the ability to self-renew and initiate
tumors; these are known as cancer stem cells (CSCs) or tumor-
initiating cells (Sheridan et al., 2006). The source of CSCs is the
topic of much investigation and debate. Although several factors
contribute to the CSC state, much evidence connects EMT with
the acquisition of stem cell properties (Al-Hajj et al., 2003; Mani
et al., 2008; Santisteban et al., 2009; Wielenga et al., 1999).
Studies in breast cancer describe the EMT cells as having proper-
ties of mesenchymal stem cells that are able to differentiate to
multiple lineages (Battula et al., 2010). Thus, similar to some of
the EMTs that occur in development, cancer-associated EMTs
result in more migratory cells capable of forming new tissues,
indicating increased stemness. CSCs exhibit dynamic changes
between epithelial and mesenchymal characteristics in squamous
cell carcinoma and breast cancer: Epithelial cells are more prolif-
erative, while mesenchymal cells are more migratory (Biddle
et al., 2011; Liu et al., 2014).
In support of the concept that EMT enhances stemness, cells that
have undergone EMT form at least 10-fold more tumor spheres, a
characteristic of stem cells. Similarly, Twist inhibits CD24 expres-
sion, suggesting a direct link to the CSC phenotype (Vesuna et al.,
2009). (Human mammary stem cells have been characterized to
express high levels of CD44 and low levels of CD24 [Al-Hajj et al.,
2003; Sleeman et al., 2006].) In addition to these studies related to
breast cancer, similar association between EMT and stemness acqui-
sition has been observed in pancreatic (Wang et al., 2009; Wellner
et al., 2009), prostate (Kong et al., 2010), and colorectal cancers (Fan
et al., 2012; Hwang et al., 2011; Hwang et al., 2014). Snail initiates
epithelial dedifferentiation in colon cancer cells (De Craene et al.,
2005). Similarly, the EMT transcription factor ZEB1 dedifferentiates
cancer cells by repressing several epithelial fate determinants (Aigner
et al., 2007). In cancers including gastric (He et al., 2012), breast
(Manning et al., 1994), liver (Tsutsumi et al., 2004), and colon (Roy
et al., 2004), Snail expression correlates with the dedifferentiated
phenotype. These results suggest that differentiated cancer cells that
undergo EMT exhibit a more CSC-like phenotype; that is, cells
become less differentiated after EMT.
Not only does EMT confer stemness properties, CSCs also display
more mesenchymal properties: Breast cancer cell lines with high
CD44+/CD24- cell numbers not only show high stem/progenitor
 14 WANG AND UNTERNAEHRER
properties, but also exhibit strongly enhanced basal/mesenchymal
markers (Sheridan et al., 2006). Similar results have been observed
in nontumorigenic immortalized human mammary epithelial cells
(HMLEs) as well. After EMT is induced via ectopic expression of
either Twist or Snail (Mani et al., 2008), most of the mesenchymal-
like cells generated indeed acquire this CD44high/CD24low pattern,
indicating that HMLEs also become more stem-like after undergo-
ing EMT. The acquired stem cell properties are also observed in
functional assays. Tumor sphere–forming ability has long been
used as a way to test cells’ stemness (Dontu et al., 2003). HMLEs in
which Snail or Twist is overexpressed form >30-fold more tumor
spheres. Also, the number of tumor-initiating cells increases at least
two-fold with constitutive expression of EMT-inducing transcrip-
tion factors, Twist or Snail (Hollier et al., 2013; Mani et al., 2008;
Morel et al., 2008), indicating dedifferentiation after EMT.
Morel et al. evaluated breast cancer stem cells by examining
the expression of CD24 (Morel et al., 2008). They observed that
CD24-negative cells, which display mesenchymal properties as
opposed to the epithelial characteristics of the CD24-positive
cells, are able to grow as tumor spheres and form tumors
in vivo. The transition to the CD24-negative fate was accom-
plished by TGF-β-induced EMT (Morel et al., 2008). Accordingly,
DEVELOPMENTAL DYNAMICS
the EMT factor Snail initiates the plasticity required in tumor
cells for both migration and stemness (Baulida and García de
Herreros, 2015). Results from Morel et al. and Baulida
et al. indicate that cells dedifferentiate after EMT.
In addition, Tran et al. and Ye et al. used mouse breast cancer
reporter models to follow the endogenous activation of Snail
and demonstrated that, in primary tumors, Snail is activated fol-
lowed by the induction of EMT, and that cells with Snail activa-
tion have higher tumor-initiation capacity, another robust
measure of stemness (Tan et al., 2014; Ye et al., 2015). These
observations also contribute to our understanding of the role of
EMT in acquisition of stemness.
As mentioned previously, EMT causes an early step in cancer
cell metastasis. Thereafter, in order to colonize a new niche and
form a metastatic tumor, reversion to a more epithelial state is
required (Ocaña et al., 2012; Tsai et al., 2012). Mouse breast can-
cer cells in vivo spontaneously undergo EMT, causing them to
disseminate, after which they revert back to the epithelial state
to form metastases (Beerling et al., 2016). Although the first
EMT of the metastatic process is often associated with cells gain-
ing stemness, a subsequent MET might be the reason most
metastases are differentiated (Brabletz et al., 2001).
In summary, the literature on EMT and stemness in cancer
suggests that the process of EMT may be causative for the stem
cell phenotype.
Mechanism of EMT-induced Cancer Stemness
The mechanism by which EMT exerts these stemness effects is
not completely understood. In this review we focus on the direct
transcriptional actions of the EMT factors. Besides their protein-
coding targets, one way that EMT transcription factors act is by
regulating the expression of non-coding RNAs. MicroRNAs
(miRNAs, small non-coding RNA molecules that function in
post-transcriptional regulation of gene expression) play a pivotal
role in regulating differentiation (Lee et al., 2005; Takamizawa
et al., 2004; Wellner et al., 2009). Factors regulating miRNA
expression and processing could thus influence the balance
between stemness and differentiation (Takahashi and Yamanaka,
2015). Examples of differentiation-promoting miRNAs are the
let-7 family and miR-34. The let-7 family of miRNAs is present
in all somatic cells (Boyerinas et al., 2010), thus presenting a
barrier to stemness. Let-7’s pluripotency and oncogene targets
qualify it as a tumor suppressor (Boyerinas et al., 2010). Let-7
inhibits self-renewal genes such as Lin28, Myc, and Sall4
(Melton et al., 2010), and its expression is decreased in many
cancers (Dahiya et al., 2008; O’Hara et al., 2009; Takamizawa
et al., 2004). Mechanisms of let-7 loss are incompletely under-
stood (Wang et al., 2012).
Let-7 is also lost during the process of somatic cell reprogram-
ming, in which expression of the transcription factors Oct4,
Sox2, Klf4, and c-Myc cause differentiated cells to take on the
phenotype of pluripotent stem cells. Studies of reprogramming
provide clues to mechanisms of let-7 loss. In reprogramming,
let-7 levels decrease to allow expression of pluripotency targets,
and let-7 inhibition increases efficiency (Melton and Blelloch,
2010). Snail binds let-7 promoters; its expression increases early
in reprogramming in parallel with the decrease in let-7
(Unternaehrer et al., 2014). In pluripotent cells, levels of mature
let-7 are kept low to absent by the action of Lin28, which pre-
vents its processing (Viswanathan et al., 2008). In other con-
texts, Twist also represses let-7 transcription (Yang et al., 2012).
Thus, EMT factors are a potential source of transcriptional
repression of let-7 and other tumor-suppressor miRNAs.
Similar to let-7, miR-34 targets pluripotency factors, and its
expression is a barrier to stemness (Choi et al., 2011). As a p53 tar-
get, its expression is likely dysregulated in p53-mutated cancers
(He et al., 2007). Snail represses miR-34 expression (Siemens et al.,
2011), which may contribute to the stemness phenotype observed
in EMT cells. Besides let-7 and miR-34, many other miRNAs could
exert similar effects in establishment of the stem cell fate in cancer
(Esquela-Kerscher and Slack, 2006). In fact, the action of let-7 is
not sufficient to overcome the action of miRNAs that activate self-
renewal (called ESC-specific cell cycle [ESCC]–regulating or ESCC
miRNAs), including the miR290 cluster (Wang et al., 2008). Thus,
loss of let-7 is necessary, but not sufficient, to achieve self-renewal.
In addition, ESCC miRNAs must be expressed (Melton et al., 2010).
This illustrates the complicated path to stemness, as well as the
checks and balances that exist to protect the differentiated state.
As early as 1991, the loss of E-cadherin now known to be
associated with EMT was correlated with dedifferentiation of
tumors (Frixen et al., 1991). More recently, it has been shown
that expression of individual EMT-inducing transcription factors
brings about the stem cell state. ZEB1, a transcription factor that
induces EMT in carcinoma cells (Zhang et al., 2015), is specifi-
cally up-regulated in pancreatic carcinomas with stem-like phe-
notypes and is necessary for tumor initiation in a xenograft
model (Wellner et al., 2009). Similar to the effects of Snail on
let-7 and miR-34 noted earlier, the EMT transcription factor
ZEB1 inhibits miRNAs including miR-203, miR-200, and miR-
183, all of which down-regulate stemness factors such as Sox2,
KLF4, and BMI1 (Wellner et al., 2009). Hence, ZEB1 provides
another link between EMT and cell dedifferentiation by inhibit-
ing these miRNAs.
In ovarian cancer development, EMT in ovarian surface epithe-
lial cells is reported to result in dedifferentiation (Ahmed et al.,
2007; Schipper et al., 1991). EMT factors inhibit a variety of differ-
entiation pathways, including NF-kB, Tumor necrosis factor alpha,
β-catenin, and p53 (Pan et al., 2009; Sharabi et al., 2008; Šošic
et al., 2003). Another way EMT factors contribute to the

dedifferentiated phenotype is by preventing senescence. The life
span of somatic cells, in terms of cell division, is limited: Differenti-
ated cells senesce and cease dividing when they reach this limit.
Both stem cells and cancer cells overcome this barrier, which allows
them to proliferate indefinitely. Snail and Twist contribute to this
process by directly repressing tumor suppressors such as the cell
cycle regulator p16 (Ansieau et al., 2008; Li et al., 2018). Thus,
several molecular mechanisms contribute to EMT-driven stemness.
Clinically, it can also be inferred that EMT status links to stem-
ness. Based on gene expression profiling, Tan et al. developed a
quantitative EMT tumor–scoring system (Tan et al., 2014). This
system highlights the distinct EMT states for each cancer type. In
ovarian cancer, the EMT status is closely linked to the reported
gene expression–based molecular subtypes, where worse progno-
sis is associated with higher EMT score (Tan et al., 2013). In ovar-
ian cancer patients, prognosis is often indicated by
clinicopathological parameters, such as metastasis to distant
organs and response to chemotherapy (Gilks and Prat, 2009).
Metastasis and high chemoresistance also indicate a higher level
of pluripotency (Safa, 2016). This means that subtypes with higher
EMT scores are associated with the dedifferentiated state.
These findings support the conclusion that in cancer cells,
DEVELOPMENTAL DYNAMICS
potency or stemness is gained after cells have gone through EMT.
Resolution: EMT Results in Transient
Dedifferentiation Allowing Differentiation
Clearly, the relationship between EMT/MET and stemness is not
straightforward. EMT is not equal to dedifferentiation, and many
other factors and processes contribute to the complex picture of
cell differentiation state. However, going through the process of
EMT may increase the probability of enhanced plasticity (Nieto,
2013; Wahl and Spike, 2017) (Fig. 2C).
Recent studies suggest that cell dedifferentiation can also be
associated with cells undergoing a partial EMT, resulting in hybrid
Epithelial/Mesenchymal (E/M) phenotype (Forte et al., 2017; Jolly
et al., 2015; Liao and Yang, 2017; Thiery et al., 2009). Jolly
et al. postulated that the core EMT and stemness modules, miR-
200/ZEB and Lin28/let7, govern EMT decision making, determining
the position of the “stemness window” on the “EMT axis” (Bracken
et al., 2008; Burk et al., 2008; Yang et al., 2010). They showed that
under different conditions, different types of cells (epithelial, mesen-
chymal, or epithelial-mesenchymal hybrid) could gain stemness.
They demonstrated that a transcription factor that is associated
independently with both regulating stemness and EMT, OVOL, not
only stabilizes the hybrid E/M phenotype but is also predicted by
mathematical models to enable hybrid E/M cells to gain stemness
(Jolly et al., 2015). EMT processes thus have the potential to convert
stable, “terminally” differentiated states to metastable states, with
the associated increase in potential gene expression (Enver et al.,
2009). In support of this model, it is a subset of the E/M cells that
exhibit tumorigenesis and in vitro stem cell activity (Strauss
et al., 2011).
Although further studies are needed to test this model, the
idea of a moving “stemness window” alongside the epithelial-
mesenchymal axis provides an extremely valuable mind set for
future research. Recently, Grosse-Wilde et al. showed that both
epithelial and mesenchymal genes can be expressed in the same
cell, and that these hybrid E/M cells can be generated from either
epithelial cells undergoing partial EMT or mesenchymal cells
EMT IN DIFFERENTIATION AND DEDIFFERENTIATION 15
undergoing partial MET. In addition, compared to their progeni-
tors, these cells displayed increased self-renewal, plasticity, and
tumor sphere–forming ability (Grosse-Wilde et al., 2015). Partial
EMT results in stemness gains both in development and in cancer
(Jolly et al., 2014; Swetha et al., 2011). Accordingly, a change in
status toward either mesenchymal or epithelial phenotypes results
in a loss of stem cell characteristics (Strauss et al., 2011).
To illustrate our premise, cell fate decisions in both develop-
ment and cancer can be symbolized by Waddington’s landscape
(Fig. 1). Differentiation is symbolized by downhill movement in
the hills and valleys in this schema, dedifferentiation by uphill
movement. During or after the process of oncogenesis, termi-
nally differentiated cells (those at the bottom of the landscape)
or progenitors (partway up the hill) may be stimulated to
undergo EMT by a variety of aberrant signaling pathways. The
EMT process may nudge cells in an uphill direction, causing
them to obtain stem cell attributes such as self-renewal as they
dedifferentiate. Subsequent steps may involve MET, and as illus-
trated by cardiac development previously, multiple rounds of
EMT/MET may occur during the course of morphogenesis.
Physiological developmental decisions begin at the top of the
mountain with totipotent zygotes, which have the highest devel-
opmental potential. At the bottom, cells are fully differentiated.
At each cell division in development, cells choose between two
or more potential fates. Just as a hiker chooses to go left or right
at a fork in the trail, cells proceed into one valley or another,
representing varying developmental pathways. Each of these fate
decisions toward a particular differentiated cell type alters gene
expression patterns in ways that are necessary for the current
decision but may set up barriers for subsequent decisions. Thus,
certain decisions require “back-tracking” that can be thought of
as going uphill in Waddington’s landscape. EMT may provide
the mechanism for these dedifferentiation events.
Thus, in development, when cells undergo EMT, they ulti-
mately differentiate; but what is the status of the cells during
and immediately after EMT? EMT may result in partial dediffer-
entiation sufficient to allow for new gene expression required in
the nascent tissue types (Fig. 2C). A reprogramming frame of ref-
erence is instructive in our model, again illustrated by Wadding-
ton’s epigenetic landscape. Just as expression of the Yamanaka
factors in somatic cell reprogramming to pluripotency results in
expansion of developmental potential, EMT may accomplish a
similar (though less extensive) increase in the number of genes
available for the cell’s use (Fig. 1). This could allow expression
of genes that were turned off in a previous developmental step.
Some possibilities of ways in which EMT might accomplish this
include opening chromatin (McDonald et al., 2011), direct gene
expression effects leading to signaling changes (Cieslik et al.,
2013), and modifying post-transcriptional control of gene
expression (Shukla et al., 2015; Unternaehrer et al., 2014).
In fact, a similar paradigm is emerging in the reprogramming
field: Sequential EMT-MET is essential for the conversion from
mouse embryonic fibroblasts (MEFs) to neuronal cells (He et al.,
2017), and sequential EMT-MET occurs during human ESC (hESC)
differentiation into definitive endoderm in vitro, where Snail plays
a key role (Li et al., 2017). This evidence supports our proposal
that early EMT/MET may poise the cells in a state more suitable
for further cell fate conversion. Thus, EMT is proposed to power
the “uphill” dedifferentiation illustrated in Waddington’s land-
scape, allowing the “downhill” differentiation that happens in
subsequent steps.
 16 WANG AND UNTERNAEHRER
To illustrate one of these proposed mechanisms, as alluded to
previously, EMT transcription factors repress key differentiation-
promoting miRNAs, let-7 and miR-34 (Siemens et al., 2011;
Yang et al., 2012). Inhibition of these miRNAs results in
increased stemness. Our working hypothesis is that by antago-
nizing let-7, miR-34, and other small RNAs, EMT factors desta-
bilize the differentiated state to allow expression of stemness
factors.
When Snail and other EMT factors are expressed in develop-
ment, we propose that, in addition to allowing migration and
invasion required for morphogenesis, their repression of
differentiation-associated miRNAs allows for new gene expres-
sion of the miRNA targets. A theoretical example is cardiac
development, which requires expression of the basic-helix-loop-
helix transcription factor Hand1, a let-7 target, as early as
embryonic day 7.5–9.5 (Roche et al., 2013). By week three
(in the human), when cardiac development is progressing (Roche
et al., 2013), let-7 is expressed at high levels (Schulman, 2005),
and in fact let-7 plays an important role in germ layer specifica-
tion (Colas et al., 2012). Consistent with its role as a heterochro-
nic gene that regulates cell fate choices, let-7 is closely regulated
to control developmental decisions (Hammell et al., 2009). We
DEVELOPMENTAL DYNAMICS
postulate that continued expression of let-7 in cardiac progeni-
tors would prevent expression of Hand1. Accordingly, Snail
knockout embryoid bodies express Hand1 and other cardiac
genes at lower levels (Lin et al., 2014). During the developmental
of EMT(s) leading to cardiac fate, the action of Snail to derepress
Hand1 (via let-7 repression) would enable cardiac
differentiation.
Other stemness genes that are let-7 targets are re-expressed
during development, implying the necessity for repeated cycles
of let-7 repression. An example is Sall4, a pluripotency factor
that is also important for limb, neural tube, heart, and gonad
development (Tatetsu et al., 2016).
Hence, we propose that the action of Snail in cancer may
bring about a reprogramming event resulting in dedifferentia-
tion: Snail repression of let-7 and miR34 (among others) allows
for derepression of pluripotency genes that usher in the stem cell
state. The resulting cancer stem cells attain self-renewal capabil-
ities and are able to differentiate to all cell types represented in
the tumor.
Perspectives/Conclusion
Although data showing roles for EMT in both differentiation and
dedifferentiation appear to be contradictory, a possible explana-
tion that could harmonize the evidence on both sides emphasizes
the plasticity gained by cells undergoing EMT.
The fact that EMT occurs periodically during development as
an essential step in formation of many tissues implies that EMT
results in differentiation. We propose that developmental EMTs,
rather than directly resulting in cellular differentiation, allow
partial dedifferentiation, enabling the next round of differentia-
tion to occur (Battula et al., 2010). In a pathological analogy,
cancer-associated EMTs result in an increase in stemness, allow-
ing cancer stem cells to self-renew and differentiate to all cell
types represented in the tumor (Polyak and Weinberg, 2009). The
process of EMT may be a critical step in acquisition of stemness
properties seen in cancer stem cells.
Evidence is mounting that partial EMTs resulting in hybrid
cells endow cells with stem cell properties. Further studies
characterizing and defining the hybrid state will doubtless pro-
vide useful insights regarding regulation and possible interven-
tion points.
In this review, we examine the possibility that, although the
overall trajectory of developmental processes is toward differen-
tiation, cells partially and transiently dedifferentiate when they
undergo EMT. This loss of differentiation represents an opportu-
nity for adjusting gene expression programs and allows for new
gene expression required for formation of new tissue types in
development. Similarly, the transient loss of differentiation seen
in cancer-associated EMTs provides a window of opportunity for
acquisition of a stem cell phenotype. Thus, tissue stem cells or
differentiated cells in which EMT transcription factors are active
might simultaneously receive signals leading to loss of key fac-
tors maintaining differentiation. We propose a mechanism
whereby factors intrinsic to EMT might also influence differenti-
ation status.
Acknowledgements
This work was supported by a Grant to Promote Collaboration
and Translation from Loma Linda University (LLU), by LLU start-
up funding, and by the Center for Health Disparities and Molec-
ular Medicine at LLU. We thank Carlotta Glackin, Nozomi Hojo,
and Sang Nguyen for thoughtful review of the article; Mindy
Lombere for generous help with illustrations; and members of
the Glackin and Unternaehrer labs for insightful discussions.
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