Pharmacology and Therapeutics 182 (2018) 80–94

Contents lists available at ScienceDirect

Pharmacology and Therapeutics

journal homepage: www.elsevier.com/locate/pharmthera

Associate editor: B. Teicher

EMT: Mechanisms and therapeutic implications T

a,b a,b a,c a,c,⁎
Mohini Singh , Nicolas Yelle , Chitra Venugopal , Sheila K. Singh
a
McMaster Stem Cell and Cancer Research Institute, McMaster University, Hamilton, Ontario, L8S 4K1, Canada
b
Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada
c
Department of Surgery, Faculty of Health Sciences, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada

A R T I C L E I N F O A B S T R A C T

Keywords: Metastasis, the dissemination of cancer cells from primary tumors, represents a major hurdle in the treatment of
Epithelial-mesenchymal transition cancer. The epithelial-mesenchymal transition (EMT) has been studied in normal mammalian development for
EMT decades, and it has been proposed as a critical mechanism during cancer progression and metastasis. EMT is
MET tightly regulated by several internal and external cues that orchestrate the shifting from an epithelial-like
Metastasis
phenotype into a mesenchymal phenotype, relying on a delicate balance between these two stages to promote
CSC
metastatic development. EMT is thought to be induced in a subset of metastatic cancer stem cells (MCSCs),
MCSCs
CTC bestowing this population with the ability to spread throughout the body and contributing to therapy resistance.
The EMT pathway is of increasing interest as a novel therapeutic avenue in the treatment of cancer, and could be
targeted to prevent tumor cell dissemination in early stage patients or to eradicate existing metastatic cells in
advanced stages. In this review, we describe the sequence of events and defining mechanisms that take place
during EMT, and how these interactions drive cancer cell progression into metastasis. We summarize clinical
interventions focused on targeting various aspects of EMT and their contribution to preventing cancer dis-
semination.

1. Introduction into EMT-MET to explain tissue expansion and cell proliferation in mammals (Eberth,
1870; Kalluri & Weinberg, 2009). The core understanding of this con-
The epithelial-mesenchymal transition (EMT) is a fundamental cept was that a single cell, or the fertilized egg, will give rise to all other
process of development and disease progression (Nakaya & Sheng, cells in the body. Throughout development, cells will differentiate to
2013). It is comprised of a series of intricate biological and biochemical assume diverse states (Kalluri & Weinberg, 2009; Lim & Thiery, 2012),
changes that cause a cell to lose its differentiated epithelial-like state however cells can be loosely categorized into either an epithelial or
and gain a more mesenchymal-like phenotype. Each state is typically mesenchymal phenotype (Lim & Thiery, 2012; Nakaya & Sheng, 2008).
differentiated based on morphological traits, and are associated with It was assumed that at the end of development these cells remain in a
specific properties that promote their functions. Epithelial cells can be terminally differentiated state to maintain their functions
identified based on cell-cell cohesion and tissue integrity, which are (Kalluri & Weinberg, 2009). Yet, this notion has been confronted by the
essential to maintaining the body composition. Mesenchymal cells are observation of cells within terminally differentiated epithelium cycling
associated with migration and invasiveness to support epithelial tissues between epithelial or mesenchymal states, showing that phenotypic
(Hay, 2005; Shook & Keller, 2003). The transition from epithelial to states are highly dynamic and dependant on cell type and environment
mesenchymal states allows a cell to break away from its originating (Micalizzi, Farabaugh, & Ford, 2010).
tissue and travel throughout the body. In 1968, Elizabeth Hay delineated several intricate characteristics
In the early 1800's the concept of cell division was adopted as a way involved in epithelial-mesenchymal transformation through

Abbreviations: AJ, adherens junction; aPKC, atypical protein kinase; BM, basement membrane; βHLH, Beta helix-loop-helix; CSC, cancer stem cells; ECM, extracellular matrix; EMT,
epithelial mesenchymal transition; FOX, Forkhead box; GAP, GTPase-activating proteins; GDI, guanine nucleotide dissociation inhibitors; GEF, guanine nucleotide exchange factors;
GRHL, Grainyhead-like; KLF, Kruppel-like factor; LATS, Large Tumor Suppressor; Lgl, lethal giant larvae; MCSC, metastatic CSCs; MET, mesenchymal epithelial transition; MMP, Matrix
metallo-proteinases; PA, plasminogen inhibitors; PALS1, Protein associated with Lin-1; PAR, partitioning complex; PATJ, PALS1-associated tight junction; PKC, protein kinase C; PRRX,
Paired-related Homeobox; RTK, receptor tyrosine kinase; RHOA, Ras homolog gene family member A; ROCK, Rho-associated protein kinases; Scrib, scribble; TEM, transendothelial
migration; TF, transcription factors; TGF-β, transforming growth factor beta; TIC, tumor-initiating cells; TJ, tight junction; uPA, urokinase-type plasminogen activator; uPAR, urokinase-
type plasminogen activator; ZEB, Zinc finger E-box binding; ZO, zonula occludens
⁎
Corresponding author at: Department of Surgery, McMaster Children's Hospital, Scientist, McMaster Stem Cell & Cancer Research Institute, MDCL 5027, 1280 Main Street West,
Hamilton, ON L8S 4K1, Canada.
E-mail address: ssingh@mcmaster.ca (S.K. Singh).

http://dx.doi.org/10.1016/j.pharmthera.2017.08.009

Available online 20 August 2017

0163-7258/ © 2017 Elsevier Inc. All rights reserved.
M. Singh et al. Pharmacology and Therapeutics 182 (2018) 80–94

Fig. 1. Schematic of the morphological differences between epithelial- and mesenchymal-like cells.
Epithelial and mesenchymal cells can be differentiated by several features. Epithelial cells exhibit apicobasal polarity and possess cell-cell and cell-matrix attachments that are vital to
their survival. Cell polarity is established by three multiprotein complexes that interact to regulate spatial separation of apical and basal domains: the Scribble complex (Scrib-Lgl-Dgl),
the Crumbs complex (Crumbs-PATJ-PALS1) and the PAR complex (Par3-aPKC-PAR6) (Iden & Collard, 2008; Lee & Streuli, 2014). Intercellular junctions provide adhesion and com-
munication between cells, maintaining tissue stability and integrity. TJs form a belt-like structure encircling the cell, aiding the separation of apical and basolateral domains and forming
a tight seal between adjacent cells to close off the intercellular space and prevent the flow of material. AJs located below TJs and perform similarly, where they also encircle the cell and
provide intercellular adhesion, however they are relatively permeable. Gap Junctions are gaps located in the lateral cell surface and act as hydrophllic ion transport channels between
adjacent cells. Desmosomes are disc-like structures on the lateral cell surface that also provide cell adhesion and provide points of intermediate filament binding. Hemi-desmosomes are
located at the basal cell surface and connect the cell to the underlying basal lamina. The onset of EMT causes disassembly of intercellular junctions and a loss of cell polarity, switching the
cell to a mesenchymal phenotype. Substantial cytoskeletal rearrangements modify the cell shape to promote motility and invasion, signifying the first stages of metastatic dissemination.
aPKC, atypical protein kinase C; Dlg, discs large; Lgl, lethal giant larvae; neural cadherin; PALS1, protein associated with Lin-7 1; PATJ, PALS1-associated tight-junction protein; PAR,
partitioning complex.

observations of cell migration in the primitive streak of chick embryos particular common characteristics, though not all features may be
(Hay, 2005). The term “transition” has since replaced “transformation” present in every epithelial structure (Nakaya & Sheng, 2013) (Fig. 1):
in EMT to reflect the transient nature of the process, as seen with the
reverse mesenchymal-epithelial transition (MET) process i) Polarity: epithelial cells possess apical and basal polarity, with
(Kalluri & Weinberg, 2009; Lamouille, Xu, & Derynck, 2014), and the plasma membranes facing towards the lumen and facing away from
process has been identified and characterized into specific biological the lumen, respectively. Each membrane has distinct protein com-
and disease states (Thiery, Acloque, Huang, & Nieto, 2009). positions, permitting localization of different activities to specific
cellular regions and allowing directional transport of molecules (Lee
et al., 2006). Cell polarity is established and maintained by three
2. Epithelial and mesenchymal phenotypes complexes that are mutually interactive. The cytoplasmic proteins
Lethal Giant Larvae (Lgl), Discs Large (Dlg) and Scribble (Scrib)
The basic epithelial tissue is comprised of adjacent individual epi- form the Scrib complex localized to the basal domain to maintain
thelial cells cohesively arranged in single cell layers or sheets (Lee, basolateral polarity, and are considered as tumor suppressors
Dedhar, Kalluri, & Thompson, 2006). The layers can be identified by

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(Lee & Vasioukhin, 2008). The apical domain is maintained by two extracellular matrix and binding sites for intermediate filaments
complexes that localize at tight junctions: atypical protein kinase (Carraway & Carraway, 2007; Garrod & Chidgey, 2008).
(aPKC)-partitioning complex 3 and 6 that compose the PAR complex Gap junctions: Communication between cells is maintained through
and defines the boundary between basal and apical domains, and gap junction complexes, a collection of pores formed by connexion
the Crumbs complex composed of Crumbs, Protein associated with proteins that bridge between the cytoplasmic domains of adjacent cells.
Lin-1 (PALS1), and PALS1-associated tight junction (PATJ) complex These pores are water-filled, permitting the transport of small meta-
which controls the formation of the apical domain (Iden & Collard, bolites and ions, and intercellular signals between cells (Lee et al.,
2008; Lamouille et al., 2014; Nakaya & Sheng, 2013). PAR3 will 2006; Lodish et al., 2000; Mese, Richard, & White, 2007; Thiery et al.,
bind phospholipids within the plasma membrane and tight junction 2009).
proteins (JAM-A19), which allows PAR3 to recruit PAR6 and aPKC.
Subsequent association with CDC42 will induce PAR6 activation of iii) Basement-membrane (BM): Integrins are transmembrane glyco-
aPKC. Recruitment of aPKC and PAR6 by PAR3 can by inhibited by proteins, and are divided into four subgroups based on a composi-
PAR1b phosphorylation of PAR3, which causes its dissociation from tion of α and β subunits. They localize at the basal membrane that
the membrane into the cytoplasm, whereas aPKC or PAR6 phos- bind intracellular actin filaments to components of the adjacent
phorylation of PAR1b causes similar membrane dissociation extracellular matrix (ECM), such as Fibronectin, Vitronectin,
(Chatterjee & McCaffrey, 2014). PAR3 and aPKC association with Laminin, and Collagen IV. These linkages anchor the cell and forms
LgL2 promotes their trafficking to the membrane, whereas phos- the basement membrane that separates the epithelium from un-
phorylation of LgL by activated aPKC causes LgL to dissociate from derlying cells and prevents cellular invasion into either layer. This
the membrane (Rodriguez-Boulan & Macara, 2014). PAR complexes basement membrane imparts mechanical strength, counteracts
help reinforce Crumb complex activity, and PAR and Scribble tensile forces, and acts as a diffusion barrier to promote the passage
complexes are mutually antagonistic. of small molecules and gases derived from the blood (LeBleu,
ii) Cell-cell interactions: To enable integrated multi-cellular function, Macdonald, & Kalluri, 2007; Lee et al., 2006).
specialized integral membrane proteins join individual epithelial
cells together to form layers and enable intercellular interactions: The general shape of an epithelial cell is flat and polygonal. The
tight junctions, adherence junctions, desmosomes, and gap junc- cellular structure is comprised of the actin cytoskeleton, a microtubule
tions. network, and intermediate filaments that work synergistically to pro-
vide structure and mold the cell shape (Axelrod, 2006). The actin fi-
Tight junctions: Tight junctions (TJ), or zonula occludens (ZO), are laments are composed of globular-actin monomers that polymerize into
a network of branching strand-like structures located at the most topical long and thin fibres, providing the cell with both mechanical and
region of the apical surface that tether individual epithelial cells to- contractile strength and linkages to transmembrane proteins. In epi-
gether, leaving very little intercellular space and creating an inter- thelial cells these filaments display cortical organization, which is vital
cellular barrier. These structures are composed of integral transmem- to both the function of cell-surface structures and to integral membrane
brane proteins (Occludins, Claudins 1–24, junctional adhesion proteins (Tsukita & Yonemura, 1999). Intermediate filaments consist of
molecules, and various Cholera Toxin proteins), peripheral anchoring keratins that provide cellular rigidity, nuclear laminins that provide
proteins (zonula Occludens-1, -2, -3, PAR), and regulatory proteins (α- stabilization of the nuclear envelope, and Vimentins that provide me-
catenin, Cinuilin, Symplekin). TJs maintain the integrity of the epi- chanical strength. Together these filaments accept tension to maintain
thelial layers as they enclose a three-dimensional space while pre- the cell's shape and confer rigidity to the structural framework of the
venting the cells from meandering away and by controlling the para- cell, and organize the internal cellular structure and organelles. Mi-
cellular and transcellular diffusion of ions and molecules (Lee et al., crotubules are protofilaments, formed by α- and β-tubulin that are ar-
2006; Lodish et al., 2000; Nakaya & Sheng, 2013; Thiery et al., 2009; ranged into a hollow tube to support intercellular transport and motion
Yilmaz & Christofori, 2009). (Sun, Fang, Li, Chen, & Xiang, 2015). The movement of epithelial cells
Adherens junctions (aka zonula junctions): Adjoining cells are able is restricted by their shape and attachments, allowing migration of
to adhere to each other through adherens junctions (AJ), localized on epithelial sheets (en block) (Fletcher & Mullins, 2010; Lee et al., 2006;
the lateral membranes below the TJs to form belt-like structures run- Yilmaz & Christofori, 2009).
ning around the cell. These junctions are anchored to the actin and Mesenchymal cells possess characteristics that, contrary to those of
myosin filaments of the cytoskeleton via interactions with the cyto- epithelial cells, enable their migration and invasion of tissues (Fig. 3).
plasmic domain of catenins (α, β, γ), and cadherins. The transmem- They have neither uniform composition nor cellular adhesion, and
brane glycoprotein E-cadherin is considered to be the quintessential possess front-to-back polarity. Mesenchymal cells are morphologically
epithelial gene, modulating cytoskeletal organization by anchoring its irregular, displaying an elongated, spindle-like shape that allows for
cytoplasmic domain to the cytoskeleton via α-catenin and β-catenin less rigid topological functionality (Pasquier, Abu-Kaoud, Al
and securing connections between adjacent cells (Lodish et al., 2000; Thani, & Rafii, 2015; Voulgari & Pintzas, 2009). Actin filaments are ar-
Yilmaz & Christofori, 2009). Moreover, AJs maintain tissue specific in- ranged in bundles of stress fibres, and form new actin-rich membrane
teractions between cells, but their belt-like arrangement can also act as projections that promote various types of movement and sensory re-
a tension cable to control cell tension and shape (Lodish et al., 2000; ception. Additionally, keratins are downregulated and Vimentins are
Yilmaz & Christofori, 2009). upregulated to increase the strength of the cytoskeleton, making it more
Desmosomes: Desmosomes are molecular complexes clustered on flexible and less prone to damage during migration (Williams, Engler,
the cytosolic surface of the plasma membrane that bind two cells to- Slone, Galante, & Schwarzbauer, 2008).
gether via transmembrane linker proteins. Desmogelin and desmo-
collin, members of the cadherin family, span the distance between 3. Transcriptional regulation of EMT
adjacent epithelial cells to link to the desmosomal cadherins of the
adjacent cell. The cytosolic complexes are composed of the outer dense Although the precise driving events of EMT remain unknown, it is
plaque, comprising plakoglobin and plakophilin that bind the cad- apparent that EMT is context dependent, induced during the activity of
herins, and the inner dense plaque that attaches the intermediate cel- numerous signalling mechanisms and under the regulation of several
lular filaments. Desmosomes aid in mechanical support and are often pathways. Though SNAIL, TWIST, and ZEB are TFs have been identified
found in cells undergoing mechanical stress. Hemi-desmosomes are si- as master regulators of EMT, they functionally cooperate with several
milar in form to desmosomes but provide cell anchorage to the others to initiate EMT by repressing epithelial characteristics and

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Fig. 2. Summary of signalling pathways involved

in EMT induction
In cells that are undergoing EMT, several signal-
ling pathways are engaged, many of which
overlap to present an intricate network of genes,
molecules and cytokines. A limited representa-
tion of their crosstalk is presented. Dynamic re-
arrangement of the cytoskeleton is crucial to cell
mobility and migration in metastasis. TGF-β co-
operates with other signalling pathways to pro-
mote EMT in various ways. Through its canonical
pathway, TGF-β activates SMAD complexes that
will subsequently activate expression of tran-
scription factors that regulate apoptotic and cell-
cell attachment genes. In the non-canonical
pathway, TGF-β will activate PI3K/Akt signalling
to increase translation, cell size, and circumven-
tion of anoikis. Through the activation of Rho
GTPases as well as JNK, TGF-β will increase cell
junction disruption and induce cytoskeletal re-
arrangements necessary to migration and inva-
sion. The map/Erk pathway can be activated by
either with cell-cell contacts via Integrins or from
TGF-β activity, which can lead to activation of
the PI3K pathways and promotion/maintenance
of mesenchymal characteristics. Map/Erk path-
ways can similarly be activated by growth factors
acting through RTKs. Activation of WNT, Hippo
and Notch pathways lead to decreased expression
of cell attachment factors, promoting cell de-
tachment as well as preventing anoikis (Du 2016
molecules, Yilmaz for pathway image, marcucci,
Larue 2005 oncogene has best). Integrin-linked
kinase; MKK, MAPK kinase; mTORC2, mamma-
lian TOR complex 2; Notch-IC, intracellular
fragment of Notch; PTCH1, patched 1; SHH, sonic
HH; SMO, smoothened.

activating mesenchymal genes in the MCSC. For instance, several TFs mediated Wnt (Komiya & Habas, 2008; Yook, Li, Ota, Fearon, & Weiss,
bind E-cadherin E-boxes, sequences in the E-cadherin promoter region 2005), NFκB (Tsubaki et al., 2013), and Notch (Leong et al., 2007;
with a shared consensus 5′-CACCTG-3′ core, to induce epigenetic si- Sahlgren, Gustafsson, Jin, Poellinger, & Lendahl, 2008). SNAIL1 also
lencing and signify the beginning stages of EMT (Hennig, Lowrick, collaborates with other TFs in EMT, as it can interact with ETS1 to
Birchmeier, & Behrens, 1996; Lamouille et al., 2014) (Fig. 2). The ma- activate MMP expression, and can directly target and activate ZEB1 and
jority of the signalling pathways, while alone are capable of EMT in- TWIST which further activate SNAIL1 to preserve EMT activity
duction, often converge or cooperate with other pathways to regulate (Lamouille et al., 2014; Peinado, Olmeda, & Cano, 2007; Wang, Shi,
EMT-TFs and compose a complex nonlinear network of EMT signalling Chai, Ying, & Zhou, 2013).
(Lim & Thiery, 2012).

3.2. Zinc finger E-box binding (ZEB) proteins

3.1. SNAIL proteins
ZEB1 and ZEB2 proteins are zinc finger homeobox proteins that can
Of the three SNAIL proteins identified in vertebrae, SNAIL1 and behave as both transcriptional activators or repressors to induce EMT.
SNAIL2 have both been confirmed to play an integral role throughout Overexpression of these proteins of epithelial markers (i.e. cadherins),
EMT of all types. They recruit factors such as Polycomb Repressive polarity proteins (i.e. CRB2, HUGL2), gap junction proteins (i.e. con-
Complex 2 (PRC2) to coordinate histone hypermethylation and deace- nexions), and desmosomes, while simultaneously activating the ex-
tylation, which ultimately represses epithelial gene expression (ex. their pression of mesenchymal genes (i.e. Vimentin, Ncadherin) (Sanchez-
primary target E-cadherin, occludins, cadherin-16, and transcription Tillo et al., 2011; Vandewalle et al., 2005). ZEB1 has also been shown to
factor 2 (TCF2) to promote cellular detachment, while increasing ex- have the strongest correlation with EMT across several cancer tissues
pression of mesenchymal genes (N-cadherin and ZEB1) and proteases (Aigner et al., 2007). Both ZEB1 and ZEB2 possess SMAD interacting
(ex. MMP2, MMP9) to promote an invasive phenotype (Bolos et al., Domains (SID) to bind R-SMADs in the canonical TGFβ pathway,
2003; Cano et al., 2000; Lamouille et al., 2014). Several stimuli and leading to their dissociation from the co-repressor C terminal binding
pathways cooperate in the regulation of SNAIL1 and SNAIL2 in dif- protein (CtBP) and switch from repressive TF to activating TF (Postigo,
ferent cell contexts, including the canonical TGF-β (Zhang, 2003). ZEB1 can also be activated by directly by the Notch (Brabletz
Tian, & Xing, 2016) or TGF-β-induced MAPK, HGF and EGF activated et al., 2011) and Wnt (Sanchez-Tillo et al., 2015) pathways.
ERK (Han et al., 2016; Kim, Kong, Chang, Kim, & Kim, 2016), GSK3β

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3.3. Beta helix-loop-helix (βHLH) proteins 3.7. Grainyhead-like (GRHL) proteins

TWIST1 and TWIST2 belong to family of over 100 βHLH TFs, which Three GRHL proteins (GRHL1, 2, and 3) comprise the family of TFs
form homo- or heterodimers with E12 and E47 to regulate DNA binding that contribute to the regulation of EMT in wound healing, epidermal
and to inhibit the transcription of epithelial genes while inducing ex- junction development, and neural tube closure in embryonic develop-
pression of mesenchymal genes. TWIST recruits the methyltransferase ment (Wang & Samakovlis, 2012). GHRL2 has been identified as a
SET8 to promote the methylation of H4K20, a histone associated with general suppressor of EMT, acting through either ZEB1 repression or
the repression of E-cadherin and the activation of N-cadherin pro- inhibition of the TGF-β pathway (Cieply et al., 2012; Quan et al., 2014).
moters, which indirectly increases alpha smooth muscle actin (α-SMA) The overexpression of GRHL2 can either promote or suppress EMT by
and MMP9 expression to aid cell invasion (Cubillo et al., 2013; modulating the expression of Cldn4, Rab25, and Nkx2-1, which are
Lamouille et al., 2014; Margetts, 2012). The downregulation of E-cad- crucial genes responsible for the differentiation and upkeep of junctions
herin in turn upregulates TWIST in a feed-forward loop, and also acti- in epithelial cells (Kang, Chen, Kim, Kang, & Park, 2009).
vates SNAIL1, to maintain EMT (Yang, 2004). TWIST1 can also over-
come cellular senescence and apoptosis incurred by detachment by
binding to p53 to promote its degradation, a system that also aids 3.8. Brachyury
MCSCs to abrogate anoikis(Puisieux, Valsesia-Wittmann, & Ansieau,
2006). TWIST and E47 can be functionally inhibited upon binding with Brachyury is a TF within the T-box gene family that is responsible
inhibitor of differentiation (ID) proteins (Cubillo et al., 2013). Similar for the definition of the midline of bilateral organisms (Le Gouar,
to the SNAIL TFs, several pathways collaborate to activate TWIST Guillou, & Vervoort, 2004). Branchyury has been identified as a novel
(Lamouille et al., 2014). tumor antigen and driver of EMT. Its overexpression can activate Akt
and SNAIL1 to downregulate E-cadherin and promote invasion (Song,
Chen, Peng, Tang, & Jing, 2016). It is a direct target for the Wnt and
3.4. Kruppel-like factor (KLF) proteins FGF-mediated receptor tyrosine kinase (RTK) pathways, and regulates
the expression of Wnt3a and Wnt8 to form a positive feedback loop
Many members of the KLF family of zinc finger proteins have been (Arnold et al., 2000).
implicated in cancer progression by acting as transcriptional activators
and/or repressors for EMT genes. For instance, KLF8 binds the GT box
of the E-cadherin promoter to repress its activity, and has been iden- 3.9. Activator protein 1
tified as a vital cancer-regulating protein (Lahiri & Zhao, 2012; Wang,
Sloss, Zhang, Lee, & Cusack, 2007). Conversely, KLF4 has been shown to The activator protein 1 (AP-1) complex, also known as an early
maintain epithelial differentiation by inhibiting EMT through the tar- response transcription factor, is a heterodimer formed from the com-
geting of numerous adhesion and cytoskeletal genes and negative reg- bination of c-Jun and c-Fos. It is regulated by TNF receptor-associated
ulation of β-catenin mediated E-cadherin gene expression, and is factor 6 (TRAF6) and TGF-β receptor activated JNK/P38 pathway,
downregulated as EMT is activated (Sellak, Wu, & Lincoln, 2012; Tiwari working synergistically with the SMAD pathway to activate several
et al., 2013; Zhang et al., 2006). KLF6, a known tumor suppressor, di- other EMT-associated TFs (Liu, Han, et al., 2015; Zhang et al., 2016).
rectly transactivates the E-cadherin promoter to mediate its expression
levels, which subsequently causes subcellular localization of β-catenin
3.10. Nuclear factor-KappaB
and c-Myc (DiFeo et al., 2006).

Nuclear factor kappaB (NFκB) is a TF that can induce EMT and is

3.5. Forkhead box (FOX) proteins vital to transformed cell survival. By blocking TRAF1 and subsequent
cell death pathways, NFκB can protect cells from undergoing apoptosis
FOX TFs are a subgroup of helix-turn-helix proteins that contain a in response to chemotherapy or radiation (Orlowski & Baldwin, 2002).
winged-helix motif of 80–100 amino acids to bind DNA, not to be NFκB can also promote EMT via various pathways. Through activation
confused with βHLH genes. Several FOX TFs repress the expression of by the IGF-stimulated PI3K pathway, NFκB can induce SNAIL1 stabili-
polarity complexes and cell-cell junctional proteins, whereas a defi- zation, whereas NFκB activation through GSK3β inhibition will upre-
ciency in other FOX genes relieves repression of other factors in order to gulate SNAIL1 and ZEB1/2 activity (Bachelder, Yoon, Franci, de
promote EMT. Overexpression of FOXC1, FOXC2, and FOXQ1 promote Herreros, & Mercurio, 2005; Chua et al., 2007; Julien et al., 2007).
mesenchymal differentiation by downregulating E-cadherin and upre-
gulating Fibronectin, Vimentin, and N-cadherin (Han et al., 2015; Mani,
St Onge, Hartman, Giaever, & Roth, 2008; Myatt & Lam, 2007; Ou-Yang 3.11. Yes associated protein
et al., 2015). Conversely, FOXM1 overexpression promotes MET, in-
creasing the expression, nuclear accumulation, and activity of β-catenin Yes Associated Protein (YAP) and PDZ-binding domain (TAZ) are
(Chiu et al., 2014). transcriptional co-activators that mediate the activity of the Hippo
pathway to drive tumor initiation and metastatic progression. Under
normal conditions YAP/TAZ are phosphorylated by Large Tumor
3.6. Paired-related homeobox (PRRX) proteins Suppressor 1 and 2 (LATS1/2), resulting in their export from the nu-
cleus into the cytoplasm and degradation by a βTRP-dependent manner
PRRX1 and PRRX2 are transcription coactivators belonging to the (Brastianos et al., 2015). Dysregulation of the Hippo pathway or a lack
family of homeoboxes located in the nucleus. Recent studies have im- of cell-cell contact leaves YAP and TAZ unphosphorylated and free to
plicated PRRX genes as novel inducers of EMT, possessing opposing translocate to the nucleus to complex with transcription factors like
roles dependant on the tumor cell type. Working through the Wnt/β- TEADs and SMADs and induce the expression of genes that upregulate
catenin pathway, PRRX1 overexpression regulates the expression of E- cell proliferation, inhibit apoptosis, and promote invasiveness. Hippo
cadherin, N-cadherin, and Vimentin (Guo et al., 2015). Conversely, the has a dual role in cancer, as several of the genes involved are recognized
loss of PRRX1 and PRRX2 was found to be required for MET and tissue tumor suppressors, whereas YAP and TaZ have been identified as on-
colonization, possibly operating through TGF-β (Du et al., 2013; Ocana cogenes (Minn et al., 2005; Piccolo, Dupont, & Cordenonsi, 2014;
et al., 2012). Yoshimasu et al., 2004).

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4. EMT classifications et al., 2012). Dysregulation of this process results in the overproduction
of fibrotic ECM proteins, manifesting as scars or keloids at the wound
EMT is recognized in three biological settings responsible for dis- site, or in organs as fibrosis, which can cause impaired organ function
tinct functions. (Kothari et al., 2014; Nistico et al., 2012). During type 2 EMT, several
transcription factors (TFs), including Erk, Snail1 and Snail 2, promote
4.1. Primary/type 1: EMT during implantation, embryogenesis, and the conversion of cells to a partial and reversible EMT state, where cells
organogenesis exhibit characteristics of both epithelial and mesenchymal cells
(Barriere, Fici, Gallerani, Fabbri, & Rigaud, 2015; Yan et al., 2010).
Cells will undergo several rounds of EMT and MET during growth
and differentiation throughout the early stages of embryogenesis and 4.3. Tertiary/type 3: EMT during tumor metastasis
organogenesis. The blastocyst is a structure formed during the initial
stages of development, and is composed of an outer layer of cells, Contrary to the subtle and controlled movement observed with
termed the trophoblast, surrounding an inner cell mast and fluid-filled other EMT types, EMT in tumorigenesis is aggressive and uncontrolled.
cavity, termed the blastocoele. EMT in activated in the trophoblast The acquisition of both EMT and the reverse MET in cancer are in-
layer to facilitate cell invasion into the endometrial lining of the uterus, dicative of tumor cells undergoing metastatic dissemination and are
and promote the establishment of a functioning placenta central to cancer progression (Kalluri & Weinberg, 2009; Lee et al.,
(Yang & Weinberg, 2008). Within the blastula, the inner cell mass will 2006).
differentiate into several structures including the primitive streak, a Metastasis is the spread of tumor cells from the primary site to a new
structure that establishes bilateral symmetry and initiates gastrulation. secondary site within the body through a process involving several
The induction of EMT will cause cells to ingress and migrate along a defined stages (Singh et al., 2014). EMT is adopted early during me-
future midline and give rise to the ectoderm, mesoderm, and endoderm, tastatic progression to permit invasion and migration of metastatic cells
where each layer will pattern the body structure (Kalluri & Weinberg, to secondary sites within the body, upon which MET is then activated
2009; Lim & Thiery, 2012; Thiery et al., 2009). MET is induced in a for the cells to revert back to an epithelial state (Pasquier et al., 2015).
subset of mesenchymal cells found in the primitive streak to form epi- Of the millions of cells shed from the primary tumor approximately
thelial mesodermal organs such as the notochord (Hay, 1995; 90% of the cancer cells are capable of completing one of more of the
Yang & Weinberg, 2008). At a biochemical level, primary EMT is early steps of metastasis, and only about 2% of these cells are able to
regulated by signalling molecules in the transforming growth factor establish micro-metastatic growth. However, it has been demonstrated
beta (TGF-β) superfamily, Wnt family, and Notch family, and interplay that only a mere 0.01% of these cells are able to survive the entire
with RTK signalling (Acloque, Adams, Fishwick, Bronner- metastatic cycle to produce full macro-metastatic tumors, revealing the
Fraser, & Nieto, 2009; Kim, Yi, Kim, & Choi, 2014). inefficient nature of this complex disease (Chambers,
Migratory neural crest cells are generated from neuro-ectodermal Groom, & MacDonald, 2002; Luzzi et al., 1998). It has been theorized
epithelial cells that have undergone EMT. The cells will delaminate, that this rare population of metastatic cells is a subgroup of cancer stem
causing a loss of polarity and intercellular junctions, disrupting the cells (CSCs) (Hermann, Huber, & Heeschen, 2008; Liao, Ye, Deng,
basement membrane and allowing the cell to invade the surrounding Bian, & Ding, 2014).
tissue. This enables cells to migrate along a rostrocaudal gradient until
reaching their destination, upon which they will differentiate and give 5. Metastatic cancer stem cell (MCSC)
rise to the craniofacial features, the peripheral nervous system, and the
melanocytes (Huang & Saint-Jeannet, 2004; Lim & Thiery, 2012). An In general CSCs are immortal tumor-initiating cells (TICs), distinctly
interesting aspect of EMT in neural crest cells is their collective mi- different from the general tumor bulk. They exhibit self-renewal and
gration, where the extensive interaction between cells allows them to proliferative properties enabling initiation and maintenance of tumor
move as an organized unit (Micalizzi et al., 2010). growth, and undergo asymmetric division and differentiation to give
EMT and MET are also employed during several later phases of rise to all the other cell phenotypes within the heterogenous tumor
embryonic development, including the formation of the cardiac heart (Lobo, Shimono, Qian, & Clarke, 2007). CSCs can be identified, isolated,
valves (Runyan, 2000-2013), the skeletal muscle, and the palate and most recently targeted, by specific biomarkers in most human
(Fitchett & Hay, 1989). Post-development, the epithelial cells maintain cancers (Chen, Huang, & Chen, 2013). The increased exhibition of EMT
a tissue-specific function while the mesenchymal cells remain as sup- activity at the leading edge of primary or metastatic tumors suggests the
port (Kalluri & Weinberg, 2009; Lim & Thiery, 2012). existence of at least two different forms of CSCs, a progression-sta-
tionary subset and a motile subset (Liao et al., 2014). The stationary
4.2. Secondary/type 2: EMT during wound healing, tissue regeneration, and CSCs are active throughout primary tumor progression but are unable
organ development to escape the primary tumor borders. The motile CSCs, or metastatic
CSCs (MCSCs), are selected and primed for dissemination by receiving
During wound healing, re-epithelialization occurs in three stages: i) signals from the stroma resembling the environment of a distant organ
inflammation, to limit tissue damage through phagocytosis, ii) pro- (Baccelli & Trumpp, 2012; Brabletz et al., 2005; Oskarsson,
liferation, to form granulation tissues, promote angiogenesis, and de- Batlle, & Massague, 2014). The activation of EMT in MCSCs conveys
posit new ECM components, and iii) maturation. Inflammation is resistance to numerous tumor suppressors to aid their survival during
mediated by inflammatory and fibroblastic cells, where exposure to dissemination.
various inflammatory molecules and cytokines, mainly TGF-β and IL-1,
will cause several cell types to differentiate into myofibroblasts 6. EMT in metastatic progression
(Kothari, Mi, Zapf, & Kuo, 2014) and interact with ECM proteins in-
cluding collagens, laminins, and elastins. Upon recruitment to the The induction of EMT is one process that can lend MCSCs in-
wound site, the myofibroblasts will digest the damaged tissues through numerable specialized features to survive escaping the primary site and
the production of MMPs and ECM-degrading enzymes, before synthe- dissemination throughout the body, and the reverse process of MET can
sizing a scaffold through ECM protein deposition to promote wound enable cells to colonize the secondary environment. These transitions
healing and contraction (Kothari et al., 2014; Nistico, Bissell, & Radisky, follow several dynamic changes in morphology, cytoskeleton, adhesion,
2012). Upon completion of tissue repair, the ECM scaffold will be de- and mobility, with the ultimate goal of promoting an invasive pheno-
graded and the myofibroblasts will undergo mass apoptosis (Nistico type (Fig. 3).

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Fig. 3. Role of EMT in metastatic cell dissemination

Following the induction of EMT, MCSCs will initiate the metastatic cascade. 1) The MSC will lose adhesion to neighbouring cells and the ECM, allowing them to break away from the
tumor bulk and 2) invade the surrounding tissue. 3) To intravasate into adjacent blood or lymphatic vessels, metastatic cells initiate local neoangiogenesis, allowing the formation of
vessels possessing weak endothelial cell junctions that the cells can easily pass through as they undergo TEM. 4) Upon entering the circulation, metastatic cells will circumvent lethal
barriers such as shearing forces and host immune responses before arresting at a new secondary site. 5) The metastatic cells will undergo TEM again to extravasate and invade the BM
surrounding the vessel, where they will undergo 6) MET and enter either a dormancy state or initiate colonize the tissue (Cavallaro & Christofori, 2004; Reymond, d'Agua, & Ridley, 2013;
Singh et al., 2014). EMT, epithelial mesenchymal transition, MCSC, metastatic cancer stem cell; ECM, extracellular matrix; TEM, transendothelial migration; BM, basement membrane;
MET, mesenchymal epithelial transition.

6.1. Cell detachment Fas) with caspase-8, causing cleavage of executioner caspases (i.e.
Caspase-3). In the intrinsic pathway, BH3-only proteins (i.e. Bim, Bad,
The cell surface is drastically altered during EMT, prompting the Bik) activate Bax/Bak to promote the assembly of Bax-Bak complexes
relocalization or degradation of intercellular attachments between and and counteract the anti-apoptotic effects of Bcl-2. Both pathways result
within cells, consequently weakening contact to neighbouring cells and in the release of cytochrome-c into the cytoplasm to induce the for-
releasing the cell from the epithelium (Shankar & Nabi, 2015). During mation of the apoptosome and to activate executioner caspases
EMT, the activation of the protein kinase C (PKC) pathway or certain (Heerboth et al., 2015; Kim, Koo, Sung, Yun, & Kim, 2012; Paoli,
oncogenic mutations can cause a depression of occludin and claudin Giannoni, & Chiarugi, 2013).
expression, resulting in TJs that are “leaky” and permeable (Lamouille MCSCs have developed various methods of avoiding anoikis. They
et al., 2014; Martin & Jiang, 2009). Desmosome complexes become undergo an “integrin switch”, entailing the downregulation of one set of
disrupted and the decreasing levels of connexion compromises gap integrins while upregulating others. For instance, increased expression
junction integrity. A central hallmark of EMT is the “cadherin switch”, of integrin αvβ6 has been noted to regulate invasion and inhibit
wherein E-cadherin is downregulated or degraded by several con- apoptosis in several cancers (Bandyopadhyay & Raghavan, 2009). By
tributors, destabilizing the AJs and priming the MCSC to dissociate modulating pro- and anti-apoptotic genes, cells can exploit a pro-sur-
from its neighbours. Moreover, the upregulation of N-cadherin, a cad- vival signal by overexpression or constitutive activation of integrins.
herin of neural specificity, will cause the transitioning cell to be at- Downregulation of E-cadherin promotes cytoplasmic accumulation of
tracted to other mesenchymal types, facilitating cell migration and in- β-catenin and subsequent interaction with the MAPK pathway, and the
vasion. The loss of E-cadherin alone has also been shown to initiate upregulation of N-cadherin increases PI3K/Akt pathway activity, both
EMT (Cavallaro & Christofori, 2004; Yilmaz & Christofori, 2009). The contributing to anoikis resistance. Modifying cellular metabolism is
overall transcriptional repression of junction proteins confers a loss of another method of anoikis resistance. MCSCs activate hypoxia inducible
cell polarity, exacerbated by the repression of the polarity complexes factors (HIF) to overcome the pockets of hypoxia and anoxia created
(Lamouille et al., 2014). from rapid tumor cell proliferation, which also promotes the activation
Anoikis is the process of self-inflicted death that occurs in ancho- of TWIST and NFκB, sustaining the activity of SNAIL and MAPK
rage-dependant cells that detach from the ECM in order to prevent pathway, all to control autophagy and metabolically sustain anoikis
dysplastic cell growth (Giannoni et al., 2008). The interaction of in- resistance (Paoli et al., 2013).
tegrins maintains cell survival by influencing various kinases (i.e. FAK,
Src, ILK) and pathways (i.e. PI3K/Akt, Hippo, MAPK), where their
disruption due to inactivation or overexpression of signalling molecules 6.2. Invasion
can cause cells to detach from the basement membrane (Lamouille
et al., 2014). As cells attach to the ECM, the FAK pathway is activated Local invasion of tissue is an early step in shaping the metastatic
and triggers a signalling cascade, upregulating Bcl-2 and FLICE in- potential of cells undergoing EMT (Fig. 3, step 1). Different strategies
hibitory protein (FLIP) to maintain cell survival (Frisch & Ruoslahti, are employed by tumor cells to overcome adjacent barriers (i.e. ECM).
1997). Detachment from the ECM will cause cessation of these signals, Cells that retain their cell-cell contacts can adopt collective migration,
activating the intrinsic and extrinsic anoikis pathways. The extrinsic where a main cell will lead follower cells into narrow lines, clusters or
pathway relies on the binding and activation of death receptors (i.e. broad sheets (Friedl & Alexander, 2011; Yu et al., 2013). Collectively
migrating cells can vary between epithelial and mesenchymal

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Fig. 4. Cellular structures that aid cell migration and invasion

Actin filaments are a primary component of the cytoskeleton, residing under the plasma membrane to provide structural support and maintain tissue integrity. (A) Tumor cell invasion is
assisted by several protrusive structures such as lamellopodia, filopodia, invadopodia or membrane blebs, which are formed through the nucleation, rapid assembly and disassembly of
these actin filaments. (B) During active migration, cells will undergo mesenchymal-type movement, where the cell will first polarize and establish points of focal adhesion with the ECM
fibres, then the cell will translocate through contractions of the cell body, leaving behind the remodeled EMC. For this process, CDC42 is activated and binds to the N-WASP and IRSp53,
which will bind the actin-nucleating ARP2/3 complex to promote actin polymerization and formation of the leading edge and wave-like lamellopodia. CDC42 binding to mDIA2 also
activates nucleation of unbranched actin. CDC42 activation of PAK will activate LIMK to inhibit cofilin and increase actin turnover, inducing the formation of sensory filopodia. Activation
of Rac induces actin polymerization and the formation of lamellopodia similarly to CDC42 but operates through the WAVE complex. The Src pathway inhibits ROCK and N-WASP to
promote invadopodia and podosome formation, which are actin-rich protrusions tailored for ECM degradation (Friedl & Wolf, 2003; Heasman & Ridley, 2008). Non-apoptotic blebs are
structures utilized in amoeboid movement in passive migration. Blebs are formed by intracellular hydrostatic pressure resulting compression of the cytoskeletal network as it separates
from the plasma membrane, which pushes out on areas of weak cortical actin in the plasma membrane. This protrusion is initially devoid of actin as it expands, and is reformed as the bleb
retracts. As blebs are formed they polarize, and hydrostatic pressure will deform the nucleus and force the cell to move forward through these blebs (Friedl & Wolf, 2003; Nurnberg,
Kitzing, & Grosse, 2011). This process is initiated by GEF activation of RHO, which works through ROCK and MLC to produce actomyosin contractions (de Lucas, Bernal, Perez, San
Martin, & Galvez, 2016; Morley et al., 2014). N-WASP, Wiscott-Aldrich syndrome protein; IRSp53, insulin receptor substrate p53; mDIA2, mammalian diaphanous 2; WAVE, WASP-family
verprolin-homologous protein; RHO, Ras homolog gene family member; ROCK, Rho-associated protein kinases; GEF, guanine nucleotide exchange factors; MLC, myosin light chain.

phenotypes, dependent on the follower or or leader cells Remodelling the cellular cytoskeleton is vital to the physical ability
(Clark & Vignjevic, 2015). Cells that have lost their inter-cellular junc- of single metastatic cells to migrate and invade. In response to in-
tions can migrate individually, a primary feature of EMT that requires tracellular and extracellular cues during EMT, the actin cytoskeleton
different morphological properties. will undergo dynamic reorganization (Fig. 4). The changes in

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morphology during cell movement can be identified in stages as the cell environments.
moves: polarization, protrusion, adhesion, translocation of the front cell
body, and retraction of the rear body. The activity of the Rho family of 6.3. Intravasation
GTPases, comprised of Ras-related C3 botulinum toxin substrate 1
(Rac1), cell division control protein 42 homolog (CDC42), and Ras MCSCs are typically positioned at the tumor's leading edge, allowing
homolog gene family member A (RhoA), is controlled by guanine nu- for better access to invade the surrounding tissue and enter the blood or
cleotide exchange factors (GEF), GTPase-activating proteins (GAP), and lymphatics vessels (Kalluri & Weinberg, 2009; Liao et al., 2014;
guanine nucleotide dissociation inhibitors (GDI), which promote cy- Oskarsson et al., 2014) (Fig. 3, step 2). Once they have invaded into the
cling between GDP-active and GTP-inactive states (Ridley, 2015). Cell surrounding stroma, MCSCs will undergo transendothelial migration
contractility is regulated via two methods. GTPase loaded RhoA will (TEM), the process of disrupting the endothelial junctions in order to
activate Rho-associated protein kinases (ROCK1 and ROCK2) to de- cross the endothelial barrier and enter the circulation. For this, MCSCs
crease myosin phosphatase activity and increase myosin light chain 2 initiate neoangiogenesis to induce the formation of new blood vessels to
(MLC2) phosphorylation, and activate LIM kinase (LIMK). This signal- connect to existing vasculature. These new vessels are generally un-
ling promotes the formation of stress fibres, the inhibition of cofilin, stable when compared to established vasculature, possessing weak cell-
actin:myosin contraction filaments responsible for focal adhesion, cell cell junctions that the metastatic cell will exploit to pass through. Notch
body contractions and lagging end retractions (Raftopoulou & Hall, signalling, VEGF and TGF-β expression are vital contributors required
2004; Rodriguez-Hernandez, Cantelli, Bruce, & Sanz-Moreno, 2016). for impairing endothelial cell function (Kalluri & Weinberg, 2009).
CDC42 and Rac1 will activate p21 activated kinase (PAK), which reg- Passage through the endothelial barrier is achieved by either active
ulates focal adhesion turnover and inactivates cofilin to promote fila- or passive migration. During active migration, several enzymes are
ment treadmilling at the leading edge of cells and to generate traction secreted either by the metastatic cell or the surrounding stromal cells to
forces to control the cell's direction (Raftopoulou & Hall, 2004). Actin promote TEM and intravasation. The urokinase-type plasminogen ac-
polymerization and filament elongation will induce extension of tivator (uPA) system consists of uPA, a serine protease, and its surface
membrane protrusions focused towards the stimulus. These protrusions receptor uPAR. Serpin plasminogen inhibitors PA-1 and PA-2 irrever-
will adhere to the surrounding stroma through the binding between sibly bind uPAR to firmly inhibit uPA activity. Overproduction of uPA
integrins and ECM protein domains. Actomyosin-mediated contractions in metastasis can overcome the inhibitors, allowing uPA to mediate
at the leading edge will pull the cell forward, with subsequent de- ECM remodelling by proteolysis, cell signalling, survival, and angio-
tachment of the trailing edge from the stroma (Sun et al., 2015). genesis (Brooks, Lomax-Browne, Carter, Kinch, & Hall, 2010).
During single cell migration two different morphologies can be Cathepsins are matrix proteases that alone can degrade BM and
adopted: mesenchymal and amoeboid (Fig. 4B). In mesenchymal mi- ECM components, and also activate uPA. Interactions with cystatins,
gration, cells appear thin and fibroblastic in shape and display Rac- their endogenous inhibitors, regulates the proteolytic activity of ca-
induced actin protrusions. Amoeboid migration is characterized by thepsins, however variation with inhibitor to cathepsin ratios due to
rounder or irregularly shaped cells that produce blebbing protrusions expression or distribution promotes the functional roles of cathepsins in
for migration (Rodriguez-Hernandez et al., 2016). The Rho-GTPase EMT (Brooks et al., 2010).
pathway is a mutually inhibitory network between Rac1 and RhoA. Matrix metallo-proteinases (MMP) are a family of calcium-de-
High levels of Rac1 inhibits Rho and ROCK signalling, suppressing pendant enzymes composed of endopeptidases, including stromelysins
amoeboid movement and promoting the cell elongation and actin-rich (MMP-3, -10, -11, 12), collagenases (MMP-1, -8, -13, -18), matrilysins
protrusions seen with mesenchymal migration. Conversely, active RhoA (MMP-7, -26), membraneous (MMP-14, -15, 16, 25), and gelatinases
and ROCK support amoeboid migration (Rodriguez-Hernandez et al., (MMP-2, -9) (Oyama et al., 2007). Under normal conditions MMPs are
2016). maintained at low levels, regulated by tissue inhibitors of metallopro-
Invading cells exhibit numerous types of plasma membrane pro- teinases (TIMPS), and are upregulated during stages of rapid tissue
trusions that aid in movement: Lamellipodia, filopodia, invadopodia, remodelling. During EMT, plasmin initiates cleavage of the inactive
podosomes, all commonly used in mesenchymal migration, and blebs proproteins into their active forms, which will then cleave cell-surface
that are associated with amoeboid migration. Lamellipodia are flat, proteins and degrade BM and ECM components, allowing metastatic
wide, wave-like protrusions found at the leading edge of the cell, arising cells to pass between adjoining cells and the basement membrane
from Rac1 stimulated actin nucleation of the plasma membrane. These (Verma & Hansch, 2007). Cleavage of E-cadherin, a chief substrate of
protrusions are used by the cell to crawl forwards (El-Sibai et al., 2008). MMPs, produces a soluble E-cadherin (sECAD) fragment that induces
Filopodia, induced by CDC42 activity, are thin cytoplasmic spikes ex- EMT in a paracrine manner through EGFR signalling
tending from the edge of lamellipodia that contain receptors for diverse (David & Rajasekaran, 2012). MMPs also cause the release of growth
signalling molecules that help navigate the cell during chemotaxis by factors from the degraded ECM, which help to promote neoangiogenesis
detecting chemotropic cues (Oh, Knelson, Blobe, & Mythreye, 2013). (Brooks et al., 2010).
Podosomes and invadopodia are cone-like actin-rich protrusions, loca- Passive migration is typical of amoeboid invasion, where cells are
lized at the front border of the cell, also initiated by CDC42 activity. able to slide through barriers without proteolytic activity. The con-
Blebs are bulky, round protrusions formed through hydrostatic pressure tractile actin:myosin core networks are vital to producing the motile
from Rho-ROCK activity. Intracellular pressure produces spherical forces. High RhoA/ROCK activity maintains cellular contractility to a
membrane ruptures caused by the detachment or breakage of the ac- degree that produces major cell deformability, allowing the cell to ra-
tin:myosin network. Consequently, the driving force of blebs is the in- dically adjust its cytoskeleton to squeeze between and around inter-
flow of cytoplasm as opposed to actin polymerization seen with other cellular spaces (Friedl & Wolf, 2003).
protrusions. Blebs possess a highly dynamic and rapid life cycle – the
blebs expand, are static for a short period, and then retract into the 6.4. Surviving in circulation
plasma membrane (Fackler & Grosse, 2008; Pankova, Rosel,
Novotny, & Brabek, 2010). MCSCs face several lethal barriers upon entrance into the circula-
All of these protrusions enable cell motility by forming attachment tion, and unlike the majority of tumor cells that succumb to these
sites between the cell and ECM (Machesky, 2008). The morphological barriers, MCSCs have adapted unique defenses (Fig. 3, step 3). The cells
adaptations in mesenchymal cells results in movement that are dis- undergo tumor cell-induced platelet aggregation by expressing
tinctly dynamic; migrating cells fluctuate between different character- thrombin, which binds coagulation factors on platelets and to form
istics in order to maintain their motility and to adapt to changing embolus aggregates and protect the metastatic cell from immune-

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surveillance and reducing the cellular stress experienced from the he- Gilja, Somarelli, & Levine, 2017), and modelling such a transiency is
modynamic shear forces (Palumbo et al., 2007). Platelets are stimulated difficult. Most studies explore a functional role of EMT in vitro, em-
by tumor cell-induced platelet aggregation (TCIPA) to secrete α-gran- ploying loss-of-function and gain-of-function methods. Fischer et al.
ules containing TGF-β and PDGF. TGF-β will then activate the SMAD developed a mesenchymal-specific Cre-mediated fluorescent marker
signalling pathways, whereas PDGF will activate the Notch pathway, switch system to track EMT in spontaneous breast-to-lung metastasis,
both resulting in the maintenance of mesenchymal properties (Labelle, where they determined that, though a small number of cells within a
Begum, & Hynes, 2011; Lou et al., 2015). Metastatic cells can also travel primary epithelial tumor do undergo EMT, the lung metastases con-
in clusters, formed by either collectively migrating cells or individual sisted of non-EMT cells (Fischer et al., 2015). Similar results were found
cells that aggregate within the circulation. The metastasizing clusters or in pancreatic cancer, through a genetically engineered mouse model of
microemboli provide similar protection as the platelet shields but also pancreatic ductal adenocarcinoma with a SNAIL1 or TWIST1 deletion
an added benefit of avoiding anoikis (Liotta, Saidel, & Kleinerman, (Zheng et al., 2015). Correspondingly, other studies leads to the idea
1976). Recent studies have provided evidence for these clusters to have that tumor cells may not be dependent on obvious upregulation of EMT
superior survival and metastatic potential over single cells, as outlined markers to become metastatic (Krebs et al., 2017; Spaderna et al., 2008;
in a recent review by Hong, Fang, & Zhang (2016). Yang et al., 2004). As such, type III EMT has been characterized to be
more of a spectrum of phenotypes, as opposed to characteristic terminal
6.5. Arrest and extravasation transition states seen in type I and II EMT, where these metastatic cells
may complete only partial transitions of EMT (Lambert,
MCSCs will localize in the vasculature of distant organs via a Pattabiraman, & Weinberg, 2017). This partial transition may allow
combination of homing mechanisms and circulation routes (Fig. 3, step metastatic cells to maintain their tumor-initiating potential, as a full
4–5). Studies have shown that circulating metastatic cells will slow transition to a mesenchymal phenotype induced through over expres-
down in capillaries of a size similar to the cell itself, often rolling along sion of EMT-TFs has been seen to impact engraftment and colonization
the endothelium before arresting. E-selectin and the expression of its (Celia-Terrassa et al., 2012; Ocana et al., 2012; Ruscetti, Quach,
corresponding ligands on MCSCs contribute to migration along the Dadashian, Mulholland, & Wu, 2015). Accordingly, EMT appears to be a
endothelium. The MCSC's anchorage to the endothelium is mediated by case-dependent process, being vital in some and more of a permissive
the binding of integrins, CD44, and MUC1, expressed on the metastatic nature in others.
cell, to selectins, intercellular adhesion molecule (ICAM1), and galectin
3, expressed on endothelial cells (Stoletov et al., 2010). MCSCs will 7. Circulating tumor cells
then commence TEM by secreting factors that activate signalling
pathways to induce openings of the endothelial cell junctions. For in- Circulating tumor cells (CTCs) are metastatic cells that have dis-
stance, as seen with cancer cell detachment in early EMT, secretion of seminated into the circulation but have not yet established secondary
VEGF or TGF-β1 by the metastatic cell disturbs the VE-cadherin-β-ca- tumor growth, and are thought to be the precursor “seeds” of metastatic
tenin complex between endothelial cells (Miles, Pruitt, van development. The measurement and characterization CTCs has high
Golen, & Cooper, 2008), allowing them to undergo cytoskeletal re- prognostic significance and offers insight into the progression of the
arrangements similar to intravasation to invade the ECM (Reymond disease, relaying information on prognosis, relapse and possible drug
et al., 2013). resistance prior to any treatment, potentially directing a different,
possibly personalized, therapeutic treatment as compared to patients
6.6. Colonization with no CTC indicators. Detection of CTCs in primary or non-metastatic
cancers has been linked to poor patient prognosis, and hundreds of
MET is the process of reversing changes incurred during EMT, clinical trials are currently utilizing CTCs as indicators for cancer pro-
where the metastatic cell will regain its epithelial characteristics and gression and survival, with varying success (Hay, 2005; Zhao et al.,
enable it to adjust to and populate the new secondary environment 2017). It has been estimated that CTCs comprise ≤ 1 of 105–106 per-
(Fig. 2, step 6). Relatively little is known about the MET process that ipheral blood mononuclear cells, where typically 1–10 CTCs can be
promotes tissue colonization, but it is clear that many of the mechan- detected in 1 mL whole blood of patients with metastatic disease (Ross
isms involved are tied to EMT. Several EMT-inducing factors are et al., 1993; Zieglschmid, Hollmann, & Bocher, 2005). Several tech-
downregulated or turned off, such as TWIST1 (Tsai, Donaher, Murphy, nologies have been adapted to detect this rare CTC population from
Chau, & Yang, 2012), SNAIL1 and SNAIL2 (Olmeda, Jorda, Peinado, liquid biopsies, a non-invasive method of testing blood or fluids, but
Fabra, & Cano, 2007), enabling cells to re-activate epithelial properties often requires an enrichment step that separates out unwanted cells
such as cell-cell contacts and promote cell growth and colonization. (Millner, Linder, & Valdes, 2013). At this stage CTCs can be character-
Intriguingly, MCSCs form distant secondary tumors that histo-patho- ized through cytometric or nucleic acid techniques.
logically resemble their primary tumor counterparts, implicating MET The exhibition of EMT and stem characteristics in isolated CTCs has
and the loss of mesenchymal characteristics as vital to colonization and been studied in several cancers, and observed to promote hemato-
secondary tumor growth (Kalluri & Weinberg, 2009). The clonality of genous spread and metastatic potential of CTCs (Aktas et al., 2009; Li
the resulting metastasis is dependent on the nature of the seeding cells. et al., 2013; Liu, Zhang, et al., 2015; Papadaki et al., 2014; Togo et al.,
Single MCSCs can develop into monoclonal metastases, whereas poly- 2017). CTCs exhibiting a mesenchymal phenotype were observed to
clonal metastases can arise from MCSC cell clusters or multiple MCSCs correlate with a more aggressive and metastatically-inclined disease
seeding one area. Recent studies with breast (Cheung, et al., 2016), progression (Zhao et al., 2017). Though several genes may mark EMT,
prostate (Gundem, et al., 2015), and pancreatic (Maddipati & Stanger, few are relevant as clinically identifiers for CTCs (Masuda et al., 2016)
2015) cancers provide evidence for polyclonal metastases, supporting (Table 1).
the concept of cell clusters as migrating metastasis initiators Typically, epithelial markers have been utilized as reliable detectors
(Cheung & Ewald, 2016). for progression of aggressive disease and metastasis, however the loss of
such markers can lead to miscalculation of CTCs with a mesenchymal
6.7. Necessity of EMT in metastasis phenotype (Togo et al., 2017). Shifts between epithelial and mesench-
ymal marker levels are commonly utilized to indicate the occurrence of
The requirement of EMT in cells to initiate metastasis has been a EMT. The cadherin switch (E-cadherin to N-cadherin) has been thor-
longstanding source of controversy. The largest barrier to elucidating oughly studied by several groups and is a significant indicator of EMT.
EMT is it being a multidimensional nonlinear process (Jolly, Ware, E-cadherin appears to have an ambiguious role in tumor and metastasis

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Table 1 8. Therapeutic targeting of EMT markers

Select clinically relevant EMT markers.
Despite the advances in cancer research throughout the years, there
EMT marker Cancer
still remain significant challenges in the treatment of cancer.
EpCAM NSCLC (Togo et al., 2017), Breast (Riethdorf et al., 2007), Chemotherapy, the gold standard treatment option, is used alone as a
Colorectal (Cohen et al., 2008), Prostate (de Bono et al., 2008) monotherapy or in combination with surgery and/or radiotherapy, but
CK19 Breast (Xenidis et al., 2009), Colorectal (Sergeant,
is often circumvented by drug resistance, a trait of cells undergoing
Penninckx, & Topal, 2008)
HER2 Breast (Wulfing et al., 2006), Gastric (Ross, 2011) EMT (Wang et al., 2016). The past decade has led to major advance-
MUC-1 Breast (Cheng et al., 2011), Pancreatic (Wang, Chen, & Tang, ments in targeted cancer therapies, as seen with monoclonal antibodies
2014) and small molecules, and over a hundred targeted therapies have been
EGFR Breast (Serrano et al., 2014; Sethi et al., 2011; Silva et al., 2006), approved for usage with hundreds more under clinical investigation
Colon (Lankiewicz et al., 2008)
(Joosse & Pantel, 2013). Unfortunately, resistance developed from long-
term usage has arisen in many patients, making drug resistance a major
progression. It has been identified as a tumor suppressor in several factor in drug discovery (Joosse & Pantel, 2013).
circumstances (Adhikary et al., 2014; Li et al., 2016; Molina-Ortiz, Chemoresistance is a major survival tactic bestowed upon MCSCs
Bartolome, Hernandez-Varas, Colo, & Teixido, 2009), yet in others high and CTCs, enabling them to evade therapy and contribute to recurrent
expression of E-cadherin is associated with increase in stemness, en- tumor growth post-treatment (Hay, 2005). Drug resistance can be either
graftment, and tumor progression (Celia-Terrassa et al., 2012; Kobel intrinsic, such as the expression of ATP-binding cassette (ABC) trans-
et al., 2011; Querzoli et al., 2010). port proteins that regulate drug efflux, or extrinsic, as seen by the gain
SNAIL and TWIST have proven to be reliable clinically-relevant of resistance to apoptosis-inducing agents. Several EMT regulating TFs
EMT-TF markers. Breast cancer cells exhibiting high levels of SNAIL (ex. SNAIL1, SNAIL2, TWIST, FOXC2) have been shown to induce these
proteins, potentially providing a novel treatment for aggressive triple- mechanisms in metastatic cells (Abdullah & Chow, 2013; Gottesman,
negative breast cancers (Micalizzi et al., 2010). TWIST1 and TWIST2 Fojo, & Bates, 2002). As CTCs have been noted as a preliminary stage to
are barely detectable in normal adult tissues, as opposed to their over- metastatic development, their particular identifying EMT character-
expression in multiple cancer types. Of the many factors that lead to the istics are emerging as novel therapeutic targets in cases of cancer
deregulation of several EMT pathways, the key identifiers of EMT are chemoresistence over typical neoadjuvant therapies (Caixeiro et al.,
members of the TGFβ and growth factor (ex. IGF, EGF, VEGF, HGF) 2014; Elaskalani, Razak, Falasca, & Metharom, 2017; Hay, 2005)
families (Divella et al., 2013; Lamouille et al., 2014; Masuda et al., (Table 2).
2016). The status of human epidermal growth factor (HER) and es- Unfortunately, targeting EMT effectors faces difficulties in reaching
trogen receptor (ER) have especially played a significant role in de- clinical settings due to the transiency associated with each phase and
termining the progression of breast cancer, making them excellent EMT functions dependent on the state of metastatic progression (Pasquier
indicators (Masuda et al., 2016). et al., 2015). Targeting of a single EMT receptor is challenging due to
Tan et al. have generated a universal EMT scoring method that the redundant nature of several EMT pathways, whereas focusing on
shows promise as an accurate tool for the measurement of EMT in EMT-TFs as a target could be more effective by having a broader effect.
cancer progression and treatment. Through analysis of transcriptomic What's more, EMT inhibitors are assumed to be most effective on EMT-
EMT signatures from lung, breast, ovarian, bladder, colorectal and associated outcomes as opposed to tumor growth (i.e. tumor cell pro-
gastric cancers, the study showed that their scoring method correlated liferation or survival), suggesting that EMT inhibitors will need to uti-
with previously published cancer-specific EMT signatures (Tan et al., lized in conjunction with established anti-tumor drugs (Elaskalani et al.,
2014). On ongoing study being conducted by Gibbons and Creighton is 2017).
surveying a 16-gene canonical EMT signature in a TCGA pan-cancer
cohort, where they have identified a correlation of several of the EMT- 9. Conclusion
related genes with worse patient outcome as well as potential novel
EMT regulators (Gibbons & Creighton, 2017). Despite of significant progress made in the development of efficient
therapeutics targeting several EMT aspects, serious challenges impede
further advancements. The plastic nature of EMT in metastasis increases

Table 2
Select inhibitors of EMT markers in clinical phase trials.

Targeting therapy Target Stage/phase Cancer

ADH-1 N-cadherin II Melanoma (Beasley et al., 2011)

Erlotinib EGFR III Hepatocellular (Zhu et al., 2015)
NSCLC (Ramalingam et al., 2014)
Gefitinib EGFR IV NSCLC (Douillard, Ostoros, et al., 2014)
Panitumumab EGFR III Colorectal (Douillard, Siena, et al., 2014)
Sorafinib TGF-β1, VEGFR, PDGFR, Raf III Hepatocellular (Cainap et al., 2015)
Renal (Motzer et al., 2014)
Dasatinib Src/FAK III NSCLC (Kelley et al., 2017)
Batimastat MMP I Hepatocellular (Xiao & Wang, 2014)
Tanomastat MMP III Ovarian (Hirte et al., 2006)
Pictilisib PI3K II Breast (Schmid et al., 2016)
Disulfiram ERK pathway IIb Breast (Nechushtan et al., 2015)
Fresolimumab TGFβ I Melanoma (Morris et al., 2014)
Renal (Morris et al., 2014)
Metformin ZEB1, SNAIL2, TWIST, Vimentin II Pancreatic (Kordes et al., 2015)
GI-6301 Brachyury I Advanced carcinomas (Heery et al., 2015)
anti-EpCAM immunotoxin EpCAM I Epithelial carcinomas (Andersson et al., 2015)
Catumaxomab EpCAM I Epithelial carcinomas (Mau-Sorensen et al., 2015)

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M. Singh et al. Pharmacology and Therapeutics 182 (2018) 80–94

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