Download Regulatory networks defining EMT during cancer initiation and

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Regulatory networks defining
EMT during cancer initiation and
progression
Bram De Craene and Geert Berx
Abstract | Epithelial to mesenchymal transition (EMT) is essential for driving plasticity during
development, but is an unintentional behaviour of cells during cancer progression. The
EMT-associated reprogramming of cells not only suggests that fundamental changes may
occur to several regulatory networks but also that an intimate interplay exists between them.
Disturbance of a controlled epithelial balance is triggered by altering several layers
of regulation, including the transcriptional and translational machinery, expression of
non-coding RNAs, alternative splicing and protein stability.
E‑cadherin
This protein is a classical
cadherin. Cadherin cytoplasmic
domains interact with a
catenin-based complex, which
couples to the actin
cytoskeleton and regulates
adhesion-dependent signalling.
Loss of function may contribute
to cancer progression by
increasing proliferation,
invasion and/or metastasis.
Department of Biomedical
Molecular Biology, Ghent
University, Technologiepark
927, 9052 Zwijnaarde,
Belgium; and Unit of
Molecular and Cellular
Oncology, Department for
Molecular Biomedical
Research, VIB, 9052 Ghent,
Belgium.
Correspondence to G.B. e‑mail: Geert.Berx@dmbr.
VIB-UGent.be
doi:10.1038/nrc3447
During epithelial to mesenchymal transition (EMT) epithelial cells are converted to migratory and invasive cells.
This process has been considered to be a fundamental
event in morphogenesis as it is intimately involved in
the generation of tissues and organs during embryogenesis of both vertebrates and invertebrates1. A similar
process is recapitulated during wound healing, a classical example of a process in adulthood in which EMT
is important. It has now been more than 10 years since
EMT was first demonstrated to be closely related to cancer progression2,3 (BOX 1). The ability of the transcription factor SNAI1 to directly repress E-cadherin has been
considered dogma since its initial observation4,5, and this
conviction has been reinforced over the years. The list of
potent EMT-inducing transcription factors (EMT-TFs)
has been growing ever since 6, revealing regulatory
networks that are often described as signalling pathways converting stimuli to transcriptional reprogramming 7,8. Endogenous expression of EMT-TFs has been
found in various tissues and is not necessarily coupled
to the dedifferentiation of tumour cells. Therefore, their
actual role is broader than the name EMT‑TF suggests.
A thorough understanding of their molecular function
can increase our knowledge of their specific contribution
in the context of cancer. Indeed, EMT‑TFs have been
involved not only in migration and invasion but also in
the suppression of senescence and apoptosis, attenuation
of cell-cycle progression and resistance to radiotherapy
and chemotherapy 1,9,10 (FIG. 1). In addition, expression
of SNAIL family members, such as SNAI1 and SNAI2
(also known as SLUG), has been observed in several
stem-cell compartments, such as those of melanocytes11,
haematopoietic cells12 and the epithelial compartment of
the gut 13. SNAIL transcription factors probably actively
participate in the maintenance of plasticity during stemcell homeostasis or exploit the plasticity during stem-cell
expansion and differentiation, as has also been suggested
for mammary-gland development 14,15. The importance
of this EMT-related reversible plasticity is clearly emphasized during embryogenesis and cancer progression, in
which the reverse process — mesenchymal to epithelial
transition (MET) — is important for final developmental
cellular differentiation and for clonal outgrowth at
metastatic sites8,16. Furthermore, the EMT–MET balance seems to fine-tune cellular destiny, as exemplified by the MET being crucial for the reprogramming
of fibroblasts towards induced pluripotent stem (iPS)
cells17,18. However, a pertinent role for EMT-TFs in the
normal homeostasis of differentiated epithelial cells
remains elusive, although they are naturally present in
various non-epithelial differentiated cell types, such as
haematopoietic cells19, lymphocytes20 and neurons21.
Importantly, these nuclear drivers of EMT seem to be
involved in both differentiation and dedifferentiation,
and this implies that their function is compatible with
both growth suppression and stimulation, depending on
the context.
For an epithelial cell to undergo a full EMT, the cell
should be permissive: the mesenchymal end-stage can be
considered the ultimate goal and will only be achieved
when all necessary component pathways of the network are activated. There is good evidence to support
the idea that dedifferentiation is triggered if the balance
between different regulatory networks is fundamentally
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At a glance
•Epithelial to mesenchymal transition (EMT) will lead to reversible reprogramming of
the cell, which is defined by fundamental changes initiated and maintained by several
regulatory circuits.
•EMT is well known to be transcriptionally regulated. Several transcription factors have
been described as potent enough to drive EMT. The often strong interconnection
between these factors forms a solid network that drives tumour progression.
•Recent evidence has linked EMT to epigenetic modifications. These are reversible,
thus emphasizing that epigenetic modifications may contribute to EMT plasticity,
which could allow cancer cells to switch back to the epithelial state on colonization at
a secondary site.
•Non-coding RNAs, and in particular microRNAs (miRNAs), are master regulators of
gene expression in many biological and pathological processes. Several miRNAs are
able to influence the cellular phenotype through the suppression of genes that are involved in controlling the epithelial and mesenchymal cell states. Moreover,
feedback loops with transcriptional regulators of EMT further define and/or maintain
a given cellular state.
•Alternative splicing of mRNA precursors leads to the formation of different proteins
from the same gene, and this directs distinct physiological functions. EMT-associated
alternative splicing events are regulated by several recently identified proteins,
adding a new layer to the complex regulation of EMT.
•More recent studies have indicated that protein levels of EMT-inducing transcription
factors are tightly controlled by additional mechanisms. A first level of control is
found at the point of translation initiation and elongation. A complex machinery
determines the stability, subcellular localization and functionality of the proteins.
Misregulation of translation and post-translational regulation may contribute to EMT
in cancer cells.
Mesenchymal to epithelial
transition
(MET). The conversion of
non-polarized and motile
mesenchymal cells into
polarized epithelial cells. MET
is typically associated with
increased E‑cadherin levels and
low cancer cell invasion and
metastasis. It is the reverse of
epithelial to mesenchymal
transition.
Vimentin
Intermediate filament found in
various non-epithelial cells,
particularly mesenchymal cells.
disturbed; this probably requires the disruption of more
than one layer (FIG. 2). Indeed, misexpression of SNAI1 or
ZEB2 in transgenic mouse models disturbs homeostasis
in some tissues, but these mice are viable and the status
of many epithelial compartments is still maintained22,23
(M. N. Tatari and G.B., unpublished observations),
hence the cell is not fully permissive and EMT is unable
to occur. EMT in epithelial cells is often observed when
the expression of an EMT‑TF is combined with an additional acquired oncogenic activity. For example, TWIST1
expression will promote tumour initiation and progression in vivo only when combined with activated RAS24.
A recent study by Rhim and colleagues25 further supports
the concept of permissiveness, using a model of pancreatic
cancer in which yellow fluorescent protein (YFP)-labelled
pancreatic epithelial cells have a mutant Kras combined
with loss of Trp53. On the one hand, induction of EMT at
an early stage is sufficient for the cells (that is, permissive
cells) to escape the primary lesions, supporting the possibility of parallel tumour progression (FIG. 1). On the other
hand, 42% of the YFP-positive cells in these tumours had
undergone EMT, supporting the existence and potency
of EMT during primary tumour progression. In fact, all
of these in vivo studies provide us with a better understanding of why EMT downstream of an EMT‑TF is much
easier to detect in vitro: cell lines are genetically aberrant,
resulting in different degrees of plasticity and allowing a
range from partial to full EMT to occur.
In the past few years it has become clear that EMT is
driven by at least four fundamental regulatory networks.
These networks are closely connected, and modulation
of any of these networks has a profound effect on every
other network. The most extensively studied network is
undoubtedly the network built around the nuclear factors of the SNAIL, ZEB and TWIST families; this network is supported by various interacting proteins, which
results in a strong transcriptional control of EMT. There
is growing evidence that three additional layers of regulation solidly support the EMT programme in parallel;
these regulatory layers are the expression of small noncoding RNAs, differential splicing, and translational and
post-translational control (which affect protein stabilization and localization). In this Review we discuss recent
insights into the control of EMT at different molecular levels and their conceptual implications for cancer
progression.
Transcriptional control of EMT
The strength of EMT is primarily dependent on the
potency of EMT-inducing transcription factors that are
capable of triggering cellular reprogramming. Overall,
proteins that are involved in transcriptional control
contribute to the best characterized regulatory network
during EMT (FIG. 2). After the initial observation of a
direct interaction of SNAI1 with the CDH1 promoter
(which encodes E‑cadherin)4,5, many other transcriptional repressors such as SNAI226, ZEB1 and ZEB2
(REFS 27,28), E47 (also known as TCF3) 29, Krüppellike factor 8 (KLF8)30 and Brachyury 31 were identified
(TABLE 1). The power of these factors relies not only on
the direct repression of E‑cadherin, but also on the
simultaneous repression of the transcription of several
other junctional proteins, including claudins and desmosomes, which facilitates the general dedifferentiation
programme32–34. The endogenous presence or the forced
expression of these EMT-TFs in cancer cells have been
linked with the loss of E‑cadherin and the gain of vimentin,
and these observations are the most commonly used to
support the importance of EMT in experimental situations. On the basis of these criteria the list of EMT-TFs
was further extended to include TWIST1 (REF. 35), forkhead box protein C2 (FOXC2)36, goosecoid37, E2‑2 (also
known as TCF4)38, homeobox protein SIX1 (REF. 39) and
paired mesoderm homeobox protein 1 (PRRX1)40. These
transcription factors seem to trigger EMT without direct
binding to the CDH1 promoter. The variety of different factors and associated functions exemplifies the
different degrees of dedifferentiation that may be
expected, and probably explains the tendency of
researchers to speak about partial EMT when dediff­
erentiation is not fully completed. Remarkably, these
EMT-TFs are exclusively associated with promoting
the mesenchymal phenotype, supporting the idea that
the epithelial phenotype is a default status41. However,
it seems that the study of the transcriptional machinery
on the epithelial side may have just been lagging behind,
as it is now becoming clear that epithelium-specific
transcription factors such as grainyhead-like protein 2
homologue (GRHL2) and the ETS-related transcription
factors ELF3 and ELF5 are downregulated during EMT
and actively drive MET when overexpressed in mesenchymal cells32,42,43. These findings seem to support
the alternative concept of a tightly controlled balance
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Polycomb group proteins
Proteins first described in
Drosophila melanogaster that
are required for normal
development. They work in
multi-protein complexes, called
Polycomb repressive
complexes, that establish
regions of chromatin in which
gene expression is repressed.
Deamination of
trimethylated H3K4
Methylation of lysine 4 (K4) in
histone H3 has been linked to
active transcription and is
removed by LSD1 and the
Jumonji C domain-containing
proteins by amino oxidation or
hydroxylation, respectively.
The deamination catalysed by
LOXL2 is a novel chemical
mechanism for H3K4me3
modification.
Transforming growth
factor-β
(TGFβ). TGFβ family members
are potent extracellular factors
that can initiate and maintain
EMT in various biological
systems. Adding TGFβ to
epithelial cells in culture is a
convenient way to induce EMT
in various epithelial cells.
between epithelial and mesenchymal status, but they still
leave room for partial EMT to occur (FIG. 2). Over the
years it has also become clear that many of the EMT-TFs
often function in the same pathway or in synergy with
each other. Remarkably, although some of them are only
mentioned sporadically in the context of EMT, others
seem to be common in the majority of the studies, in
particular the nuclear factors of the SNAIL, ZEB and
TWIST families. Their consistent association with EMT
suggests that the collection of master EMT-TFs may be
complete, and that most of the other factors may have
a facilitating function, or at least they will feed to these
master EMT-TFs, finally resulting in EMT.
One of the reasons for the high potency, and thus
abundance in the literature, of the SNAIL, ZEB and
TWIST proteins might be their close interaction with
different epigenetic modifiers, thus allowing fundamental genome-wide changes in gene expression. For
a long time, epigenetic changes during EMT were primarily studied on the CDH1 promoter. DNA methylation on CpG dinucleotides in regulatory sequences is a
typical mechanism for silencing tumour suppressors in
cancerous cells. CDH1 promoter methylation has been
recognized as part of the programme that results in
EMT44,45, and has been associated with SNAI1 expression46. Notably, DNA methyltransferase 1 (DNMT1)
potentially modulates CDH1 expression in a methylation-independent context. This could happen by direct
interaction with SNAI1 through the amino‑terminal
region, which would thereby prevent the association
of this transcriptional repressor with the CDH1 promoter 47. Epigenetic modifications also involve histone
modifications; in particular, deacetylation and specific
Box 1 | EMT and cancer progression: a controversial issue
The clinical relevance of epithelial to mesenchymal transition (EMT) during cancer is
frequently questioned. This is mainly because evidence of a full EMT phenotype in
clinical carcinomas and metastases is generally lacking16. The failure to prove EMT on the
molecular level has been countered by the concept that EMT changes are transient and
may happen in a minority of cancer cells, which is supported by the clinical observation
that single cancer cells or small cell clusters with reduced E‑cadherin levels are present
at the invasive front139. The idea that EMT has a role in cancer has also benefited from its
link with the loss of E‑cadherin expression, which is a well-established condition for
malignant cancer progression. However, E‑cadherin loss is not always related to the
induction of EMT: for example, lobular breast cancer has genetic inactivation of CDH1
(which encodes E‑cadherin) but also has epithelial characteristics45. Furthermore, partial
or complete loss of E‑cadherin alone is usually not enough to trigger EMT-associated
transcriptional reprogramming, and can be the consequence of many EMT-independent
cellular changes that do not have a proven role in cancer dissemination140.
Evidence for a complete EMT in human cancer is clearly present in carcinosarcomas;
most other cancers have partial features of EMT and express both epithelial and
mesenchymal markers141. In addition, a transcriptomic EMT signature has been
associated with the poor prognosis of patients with basal B or claudin-low breast
tumours63,142,143. An active role of EMT during metastasis is also suggested by the finding
that the gain of expression of mesenchymal markers in circulating tumour cells of
patients with breast cancer predicts poor prognosis more accurately than only
considering epithelial cytokeratin markers144. These clinical examples showing the
relevance of EMT for human cancer are supported by many xenograft experiments and
work with genetically modified mouse cancer models. The ability to sensitively tag
cancer cells in experimental models has made it clear that EMT is associated with in vivo
epithelial tumour progression and could be involved in the seeding of distant organs
before, or in parallel with, tumour formation at the primary site25,145.
methylation or demethylation patterns are considered to lead to gene repression (reviewed in REF. 48).
It has been shown that SNAI1 induces repressive histone modifications at the CDH1 promoter through the
recruitment of many different proteins, such as histone
deacetylase 1 (HDAC1) and HDAC2 (REF. 49). Similarly,
ZEB1 cooperates with the deacetylase sirtuin 1 (SIRT1)
at the CDH1 promoter, leading to the deacetylation of
histone H3 and to reduced binding of RNA polymerase II50. Furthermore, various histone modifiers have
been linked to SNAI1 activity, such as the demethylase
LSD1 (REF. 51) and the methyltransferases EZH2, SUZ12
(REF. 52), SUV39H1 (REF. 53) and G9a (also known as
EHMT2)54. ZEB1 probably acts in a similar way to suppress CDH1 by interacting with BRG1, a subunit of the
SWI–SNF chromatin complex and a known interaction
partner of several histone-modifying enzymes55. EZH2
and Polycomb complex protein BMI1 are Polycomb group
proteins, which regulate lineage choices during development and differentiation, and both proteins cooperate with TWIST1 to repress CDH1 transcription56.
Remarkably, lysyl oxidase homologue 2 (LOXL2) has a
dual role in SNAI1‑driven CDH1 repression: although
it was initially shown to be involved in maintaining the
post-translational stability of SNAI1 (REF. 57), it also
catalyses the deamination of trimethylated H3K4 (REF. 58).
In uncontrolled situations such as cancer it seems
reasonable to accept that EMT-TFs will interact with
many of the available cofactors in the deregulated cell,
facilitating EMT.
Given the variety of potential modifications at the
CDH1 locus, genome-wide epigenetic reprogramming
during EMT has recently gained attention, and this has
been demonstrated by several genome-wide studies.
During hypoxia-induced EMT, histone deacetylase 3
(HDAC3) was identified as a direct transcriptional target of hypoxia-inducible factor 1α (HIF1α). HDAC3 is
recruited to the epithelium-specific promoters, such as
CDH1 and plakoglobin, where it cooperates with SNAI1
to mediate gene repression59. HDAC3 further interacts
with hypoxia-induced WD repeat-containing protein 5
(WDR5), recruiting the histone methyltransferase complex to increase histone-specific methylation, thereby
activating mesenchymal genes such as those encoding
vimentin and N-cadherin59. Treating cultured mouse
hepatocytes with transforming growth factor-β (TGFβ)
induces EMT, and this involves a switch in the LSD1
histone methylation pattern: genome-scale mapping
showed that DNA methylation patterns were unchanged
and that chromatin changes were mainly specific to large
organized heterochromatin K9 modifications60. Using
epithelial prostate cells it was further demonstrated that
global gene expression during EMT is regulated by the net
intensity of active histone methylation minus repressive
histone methylation and DNA methylation marks61.
Intriguingly, there is no thorough understanding of
the hierarchy, if one exists, of the different EMT-TFs,
particularly because they are often studied individually
or in limited groups, but rarely altogether. One straightforward way to do this is through the comparison of gene
expression profiles in cells overexpressing the different
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Primary tumour site
Homeostasis
Normal
epithelium
SC
Tumour progression
Adenoma
Therapy
Invasive
carcinoma
Carcinoma
in situ
Recurrence
Therapeutic
CSC
BM
Metastatic site
Resistant cell
Parallel
progression
EMT
Intravasation
MET
Blood
vessel
Extravasation
Figure 1 | Role of EMT during cancer progression. In tumour cells, epithelial to mesenchymal transition
(EMT)-inducing
Nature Reviews
| Cancer
transcription factors (EMT-TFs) may primarily redefine the epithelial status of the cell, potentially — but not
necessarily — assigning stem cell (SC) characteristics to dedifferentiated tumour cells, or they may redefine resident
genetically altered stem cells to be cancer stem cells (CSCs). The dissemination of tumour cells from the solid tumour and
subsequent migration after breakdown of the basement membrane (BM) — the classical view of the role of EMT in cancer
— can only be achieved when all component pathways of the network are activated and fully parallels the process that is
seen in development: if the cancer cell has acquired the necessary genetic aberrations and receives the appropriate
signals at the tumour–host interface, the cell is ready to move towards metastasis. At this point, the active contribution of
the EMT-associated programme is probably to give survival signals and to maintain the mesenchymal status of the
metastasizing cell. It is likely that EMT also has a role in parallel progression, in which tumour cells escape early and
metastasis progresses in parallel to the primary tumour. EMT features may further promote resistance during tumour
therapy, leading to recurrence and a poor prognosis. The degree of EMT during the different steps in cancer progression
probably depends on the imbalance of several associated regulatory networks with activated oncogenic pathways.
MET, mesenchymal to epithelial transition.
EMT-TFs or in the same cells in which these EMT-TFs
are knocked down33,62,63. Unfortunately, a correct interpretation is impeded by a large number of pitfalls, such
as the availability of compatible cofactors. However,
knockdown experiments in several cancer cell lines
with the co-expression of multiple EMT-TFs have shown
that ablation of only one EMT‑TF can be sufficient to
partially or totally block EMT and metastasis in experimental models28,64–66. In this case, the loss of expression
of an EMT‑TF probably also induces the loss of a strong
anti-apoptotic signal67 or the loss of associated stem-cell
characteristics68,69. Nevertheless, the different EMT-TFs
can drive common, as well as non-redundant, pathways:
for example, loss of the newly characterized EMT‑TF
PRRX1 enhances rather than suppresses stem-cell characteristics40, and thus its loss not only promotes MET but
also allows outgrowth at the site of metastasis.
Non-coding RNAs regulate EMT
Small non-coding RNAs or microRNAs (miRNAs) are
now commonly known as potent modifiers of gene
expression that enable cells to rapidly respond to a
new fate or environment. The number of miRNAs that
has been directly or indirectly associated with EMT is
becoming as extensive as the list of EMT-associated transcription factors70. Nevertheless, there is sufficient experi­
mental evidence to define two strong recurrent anchors
in different cellular systems (TABLE 2). One is no doubt
centred on the miR‑200 family 71,72, which has five members arranged as two clusters; mir‑200a, mir‑200b and
mir‑429 on human chromosome 1, and mir‑200c
and mir‑141 on human chromosome 12, with each cluster
expressed as a polycistronic transcript 73. Expression of
the miR‑200 family is strongly associated with epithelial
differentiation, and a reciprocal feedback loop between
the miR‑200 family and the ZEB family of transcription factors tightly controls both EMT and MET71,72.
Importantly, this miRNA-mediated differentiation is
not only due to ZEB targeting but also to regulation of
the expression of accessory epigenetic machinery such
as BMI1 and SUZ12 (REFS 69,74).
SNAI1‑dependent EMT is directly controlled by
miR‑34 family members75,76. Both miR‑200 and miR‑34
have E‑boxes in their promoters, which are directly regulated by SNAIL and ZEB transcription factors76,77, but
the specificity, redundancy and/or cooperation between the
two loops are poorly characterized. However, these
loops seem to share similarity in both upstream inducers and operation modes, further creating the contours
of a hierarchical framework that defines cellular plasticity. Moreover, additional miRNAs might provide additional support for the epithelial phenotype-like miR‑101,
which maintains E‑cadherin expression by repressing
EZH2 (REF. 78).
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EMT
MET
Epigenetic
machinery:
• EZH2
• BMI1
Post-translational
control
Transcriptional
control
Differential
splicing
Non-coding
RNA regulation
• ELF3
• ELF5
• GRHL2
• SNAI1
• ZEB
• TWIST1
• ESRP1
• ESRP2
RBFOX2
• miR-200
• miR-34
• miR-101
Epithelial features
• E-cadherin
• Polarity
Post-translational
control
Transcriptional
control
Differential
splicing
Non-coding
RNA regulation
miR-9
• Vimentin
• N-cadherin
• Fibronectin
Mesenchymal features
Figure 2 | EMT is controlled by four major interconnected regulatory networks. Proteins involved in transcriptional
Reviews
Cancer
control contribute to the best characterized regulatory network during epithelial to mesenchymal Nature
transition
(EMT).|Several
EMT-inducing transcription factors, such as SNAI1, ZEB and TWIST1 have a central role in this network. Their function is
further enhanced by the close cooperation with the epigenetic machinery that enables firm repression of epithelial
features. Epithelial-specific transcription factors are far less studied in the context of EMT; they define the differentiation of
the epithelial cell. Nevertheless, the epithelial status is dominated by the presence of several non-coding RNAs defining
another strong network controlling EMT. A tight negative feedback loop is formed by the microRNAs (miRNAs) miR‑200 and
miR‑34 with several EMT transcription factors (EMT-TFs). The epithelial phenotype is further supported by miR‑101, which
promotes E‑cadherin expression. Non-coding RNAs at the mesenchymal side seem merely to support the mesenchymal
phenotype. Differential splicing on both the epithelial and the mesenchymal sides is characterized by specific splicing
factors, which determine specific epithelial or mesenchymal splice variants. Proteins involved in the different regulatory
networks are further regulated on both translational and post-translational levels, either providing more support for or
destabilizing their function. Currently, evidence for this regulatory level is almost exclusively available for EMT-TFs (solid
arrows) and is less clear for transcription factors that maintain the epithelial phenotype (dashed arrows). Given the strong
established interconnections between these regulatory layers, it is to be expected that future research will further reinforce
this picture with additional regulatory circuits being found to exist between the molecules shown in the figure. EMT is
initiated when the balance in these regulatory networks is disturbed (for example, by extracelluar signals or protein
misregulation); the degree of EMT is further defined by how far the balance is tipped in one or the other direction. ELF3,
E74‑like factor 3; ESRP1, epithelial splicing regulatory protein 1; GRHL2, Grainyhead-like 2; MET, mesenchymal to epithelial
transition; RBFOX2, RNA binding protein FOX1 homologue 2.
EMT-associated stem cell
phenotypes
Cells undergoing EMT acquire
motility and stemness
phenotypes. Both traits are
believed to form the basis for
dissemination and metastasis
of differentiated carcinomas, as
the plasticity allows permanent
adaptations to the demanding
conditions of a changing
environment.
Interestingly, the miR‑200 family has been shown to
be downregulated in normal human and mouse mammary stem and progenitor cells79, a property shared with
miR‑205 (REF. 80), another negative regulator of ZEB1 and
ZEB2 (REF. 71). Downregulation of miR‑200 members has
also been observed in malignant cancer stem cells in the
colon, breast and prostate69,79,81. Both miR‑200s and miR‑34
are under the positive control of p53 (REFS 76,82–84).
Although the underlying mechanisms for a precise role
of p53 in stem cells are far from understood, it seems to
be able to restrain stem-cell self-renewal and to impose
an asymmetric mode of division, at least in breast tissue
(reviewed in REF. 85). p53 deficiency facilitates the reprogramming of differentiated cells into iPS cells that closely
resemble embryonic stem (ES) cells. Loss of p53 function
has now been associated with the regulation of EMTassociated stem cell phenotypes and the expression status of
stem-cell markers such as BMI1, CD44 and CD133 (REFS
76,82–84). Mutant p53 will not only prevent wild-type p53
from binding on the miR‑200 promoter 82 but will also
directly repress miR‑130b, an inhibitor of ZEB1 (REF. 86).
In a number of experimental models, mutant p53, rather
then loss of p53, was found to augment the pro-migratory,
pro-invasive and pro-metastatic properties of TGFβ
in vitro and in vivo87. As such, mutant p53 may be involved
in reinforcing the strong established network around
TGFβ, ZEB transcription factors and miR‑200 (REFS 71,77),
further facilitating our understanding of why recapitulation of EMT features in a cancer context might endow
tumour cells with selected stem-cell characteristics68,
enabling tumours to be formed more efficiently.
The epigenetic regulation of the different epitheliumrelated miRs has recently received a lot of attention.
There is an inverse correlation between mir‑200c and
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Table 1 | Molecular factors involved in EMT and cancer progression: transcriptional regulation
Molecules
Function and involvement in EMT
Relevance to cancer
Refs
Transcription factors: direct binding to the CDH1 promoter
SNAI1
Zinc-finger protein, transcriptional
repressor and cellular
reprogramming
Upregulated in breast carcinoma, colon cancer, endometrial cancer, head and
neck squamous cell carcinoma, hepatocarcinoma, gastric cancer, oesophageal
adenocarcinoma, ovarian carcinoma, pancreatic carcinoma and synovial carcinoma
146,147
SNAI2
Zinc-finger protein, transcriptional
repressor and cellular
reprogramming
Upregulated in breast carcinoma, colorectal carcinoma, oesophageal squamous
cell carcinoma, gastric cancer, head and neck squamous cell carcinoma,
hepatocarcinoma, lung adenocarcinoma, malignant mesothelioma, ovarian
carcinoma and pancreatic cancer
147,148
ZEB1
Zinc-finger E‑box binding homeobox Upregulated in bladder cancer, breast cancer, colorectal cancer, endometrial
protein, transcriptional repressor and adenocarcinoma, oesophageal squamous cell carcinoma, gastric cancer, head
cellular reprogramming
and neck squamous cell carcinoma, hepatocarcinoma, leiomyosarcoma, lung
carcinoma, pancreatic cancer and prostate cancer
147
ZEB2
Zinc-finger E‑box binding homeobox
protein, transcriptional repressor
and cellular reprogramming
Upregulated in bladder cancer, breast cancer, colorectal cancer, gastric cancer,
head and neck squamous cell carcinoma, hepatocarcinoma, lung cancer, ovarian
cancer and pancreatic cancer
147
E47
Class I basic helix–loop–helix (bHLH)
factor
Upregulated in gastric cancer and prostate cancer
KLF8
Zinc-finger protein, transcriptional
repressor and activator
Upregulated in brain cancer, breast cancer, gastric cancer, liver cancer, ovarian
cancer and renal cancer
Brachyury
Transcriptional activator
Upregulated in colorectal cancer and lung cancer
149,150
151
152,153
Other transcription factors associated with EMT
TWIST1
bHLH factor
Upregulated in bladder cancer, breast cancer, colorectal cancer, oesophageal
squamous cell carcinoma, gastric cancer, head and neck squamous cell carcinoma,
hepatocarcinoma, ovarian carcinoma, pancreatic cancer and prostate cancer
FOXC2
Transcriptional activator
Upregulated in breast cancer, colorectal cancer, oesophageal squamous cell
carcinoma and lung cancer
Goosecoid
Homeobox protein
Upregulated in breast cancer
E2‑2
Class I bHLH factor
Upregulated in lung squamous cell carcinoma
SIX1
Homeobox protein
Upregulated in breast cancer, cervical cancer and ovarian cancer
PRRX1
Homeobox protein
Upregulated in breast cancer and lung cancer; correlated with lack of metastasis
147
36,
154–156
37
157
158–160
40
Accessory associated complexes and epigenetic machinery
HDAC1,
HDAC2 and
HDAC3
Histone deacetylases
Upregulated in breast cancer, colorectal cancer, gastric cancer, hepatocarcinoma,
lung cancer, pancreatic cancer, prostate cancer and renal cell cancer
161
SIRT1
NAD-dependent histone deacetylase Upregulated in colorectal cancer and skin cancer
162
EZH2
PRC2 component
Upregulated in B cell non-Hodgkin’s lymphoma, bladder cancer, breast cancer,
colon cancer, glioblastoma, Hodgkin’s lymphoma, oral cancer, liver cancer, lung
cancer, lymphoma, mantle cell lymphoma, melanoma, prostate cancer and
testicular cancer
163
SUZ12
PRC2 component
Upregulated in breast cancer, colon cancer, germinal cell-derived tumour, liver
cancer, lung cancer, mantle cell lymphoma, melanoma, and parathyroid and
pituitary adenoma
163
LSD1
H3K4 and H3K9 demethylase and
epigenetic regulator
Upregulated in breast cancer, bladder cancer, colorectal cancer, lung cancer,
prostate cancer and sarcoma
SUV39H1
Histone methyltransferase
Upregulated in colorectal cancer
170
G9a
Histone methyltransferase
Upregulated in lung cancer
171
BMI1
PRC1 component
Upregulated in acute myeloid leukaemia, breast cancer, gastrointestinal tumour,
lymphoma, medulloblastoma, neuroblastoma, lung cancer, and parathyroid and
pituitary adenoma
163
LOXL2
A lysyl oxidase
Upregulated in colorectal cancer, breast cancer, laryngeal cancer, lung cancer,
gastric cancer, oesophageal squamous cell cancer, ovarian cancer, head and neck
carcinoma, and melanoma; and upregulated in stroma associated with colorectal
cancer, pancreatic cancer, laryngeal cancer, endometrial cancer, testicular
seminoma, hepatocarcinoma and renal clear cell cancer
164–169
172–174
EMT, epithelial to mesenchymal transition; EZH2, enhancer of zeste 2; FOXC2, Forkhead box C2; HDAC, histone deacetylase; KLF8, Krüppel-like factor 8; LOXL2,
lysyl oxidase-like 2; LSD1, lysine-specific demethylase 1; PRC2, Polycomb repressor complex 2; PRRX1, paired-related homeobox gene 1; SIRT1, sirtuin 1; SUV39H1,
suppressor of variegation 3–9 homologue 1; SUZ12, suppressor of zeste 12.
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mir‑141 promoter methylation status and expression in
breast and prostate cancer cell lines, which is accompanied by permissive and repressive histone modifications
at this promoter corresponding to an epithelial and mesenchymal status, respectively 88. Promoter hypermethy­
lation of mir‑205 has also been detected in prostate
cancer 89. Epigenetic silencing of the mir‑200 family and
mir‑205 has thus been associated with early transformation of epithelial cells90. In colorectal cancer, miR‑200
expression is lower in the primary tumour, particularly
at the invasive front 91,92, whereas higher expression is
again seen in corresponding metastases, associated with
hypomethylation of the promoter region92. Consistent
with this, it was shown that overexpression of miR‑200
in weakly metastatic breast cancer cells suppresses
SEC23A‑mediated secretion, and that this promotes
further metastatic colonization of these cells in distant
organs in mice, suggesting that the role of the miR‑200
family in metastasis not only relies on promoting MET
but also on altering other cellular properties such as the
secretome, which can influence the microenvironment
to permit metastasis93.
The miR‑200 and miR‑34 family members are
strongholds of the epithelial and differentiated phenotype, and thus are considered to be tumour suppressive
miRNAs, whereas there is also evidence for miRNAs
promoting the transition to a mesenchymal phenotype
(TABLE 2). However, the expression pattern for many — if
not all — miRNAs is often cell-context specific. miR‑9
targets CDH1 directly, but it does not necessarily induce
EMT94. Similarly, increased miR‑92a expression will
reduce CDH1 expression, resulting in the promotion of
carcinoma cell motility and invasiveness95. It is likely that
these miRNAs that are associated with the mesenchymal phenotype are downstream effectors fine-tuning the
general cellular reprogramming. This was also shown for
miR‑99a and miR‑99b: although their inhibition was not
sufficient to block TGFβ-induced EMT, their expression
could contribute to migration and lower E-cadherin and
tight junction protein ZO1 expression96.
Although overwhelming evidence supports the role
of different miRNAs during EMT, there is much less
known about other classes of non-coding RNAs. We
are only beginning to understand their complexity and
nature, leaving an emerging field in RNA biology, probably with important implications for EMT. A notable
example was the description of a natural antisense transcript that is upregulated after SNAI1‑induced EMT and
prevents the processing of a large intron that is necessary for the expression of ZEB2 (REF. 97), clearly illustrating the possibility that EMT transcripts can be present
without the need to be translated into protein. This
observation may also help us to understand the recent
finding that the ZEB2 transcript functions as a competitive endogeneous RNA (ceRNA) for PTEN miRNAs.
The aberrant regulation of PTEN following ZEB2
loss can contribute to melanoma development 98. This
described role of ZEB2 as a tumour suppressor may be
counterintuitive at first glance, but it fits well with the
idea that deregulation of a transcription factor with a
potential important function in a cell type other than
the epithelium may be involved in tumour progression.
EMT-associated differential splicing
Besides the well-established transcriptional reprogramming during EMT, post-transcriptional mechanisms
such as alternative pre-mRNA splicing also provide
an additional layer of gene regulation that further
enhances EMT. The close connection between EMT
and alternative splicing events was first shown for
fibroblast growth factor receptor 2 (FGFR2), the protooncogene RON (also known as macrophage stimulating
Table 2 | Molecular factors involved in EMT and cancer progression: non-coding RNAs
Molecules
Function and
involvement in EMT
Relevance to cancer
Refs
miR‑200
family
Tumour suppressor
function
Downregulated in bladder cancer, gastric cancer, spindle cell
carcinoma of the head and neck, ovarian cancer and endometrial
carcinosarcoma
142,
175–178
Upregulated in endometrial endometrioid carcinoma, pancreatic
cancer; higher expression in distant metastasis versus primary breast
cancer; and increased levels in blood of patients with gastric cancer
93,
179–184
Downregulated in bladder cancer, melanoma, spindle cell carcinoma
of the head and neck, head and neck squamous cell carcinoma and
prostate cancer
177,178,
185–189
miR‑205
Tumour suppressor
function
miR‑34
Tumour suppressor
function
Downregulated in breast cancer, colorectal cancer, lung cancer,
ovarian cancer, pancreatic cancer, sarcoma, renal cell cancer and
urothelial cancer
191–193
miR‑101
Tumour suppressor
function
Downregulated in gastric cancer, hepatocellular carcinoma and
lung cancer
194–197
miR‑9
Tumour promotor
function
Upregulated in brain cancer, breast cancer and gastric cancer;
downregulated in breast cancer and ovarian cancer; higher
expression in distant metastasis versus primary breast cancer; lower
expression in metastatic versus primary brain tumours
179,198
Upregulated in lung cancer and endometrial cancer
190
EMT, epithelial to mesenchymal transition; miR, microRNA.
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Exon junction arrays
Microarrays constructed to
measure the expression of
alternative splice forms of a
gene. The probes used are
specific for expected or
potential splice sites of
predicted gene exons.
Cap-independent
translation
The internal ribosome entry
site (IRES) in the mRNA 5ʹ
untranslated region is used for
translation initiation.
Cap-independent mRNAs can
bypass translation inhibition
when subjected to stresses
such as hypoxia, and some
mRNAs can use both the cap
and IRES.
1 receptor (MST1R)) and CTNND1 (which encodes
p120 catenin)34,99,100, suggesting that the balance between
specific splicing isoforms is relevant, whether or not
EMT will occur. For example, the exon 11 skipping of
RON is enhanced in post-EMT cells and results in a
constitutively active tyrosine kinase receptor that confers invasive properties. The production of this alternative RON transcript is regulated by the proto-oncogene
and splicing factor serine/arginine-rich splicing factor 1
(SRSF1) the expression level of which is attenuated by
SAM68 in epithelial cells. The level and/or phosphorylation status of the splicing regulator SAM68 exerts
its effect in epithelial cells by non-productive splicing
in the 3ʹ untranslated region (UTR) of SRSF1, resulting in alternative splicing-activated nonsense-mediated
mRNA decay 99,101. Two paralogous RNA binding proteins, epithelial splicing regulatory protein 1 (ESRP1)
and ESRP2, were identified as essential promoters
of the epithelial pattern of FGFR2 splicing in a highthroughput cell-based cDNA expression screen102. These
factors also control the specific splicing of the epithelial isoforms of CTNND1, CD44 and the orthologue of
mammalian enabled MENA (also known as ENAH).
The potential functional contribution towards the
EMT phenotype of these spliced genes is nicely exemplified by CTNND1: the epithelium-specific splicing of
CTNND1 results in a shorter isoform of the protein that
promotes cell–cell adhesion by stabilizing E‑cadherin at
the plasma membrane, whereas the longer mesenchymal
isoform induced during EMT binds RHOA and attenuates its activity, resulting in enhanced cellular invasion103.
Although this demonstrates the potential potent consequences of reduced ESRP expression, it should be clear
at this stage that EMT-related differential splicing events
occur as part of a general cellular reprogramming, and
that the specific functional contributions of different
splice isoforms are not yet clear in most cases. To determine a genome-wide ESRP-regulated alternative splicing network, the functional consequence of either ESRP
knockdown in cells with an epithelial differentiation
pattern or ectopic ESRP expression in MDA‑MB‑231
cells (post-EMT) was measured using exon junction
arrays. This resulted in the discovery of more than 200
validated ESRP-enhanced or ESRP-silenced exon splicing changes104. On the basis of these well-defined splicing
events, the search for conserved sequences in the exons
and flanking introns resulted in the identification of
enriched motifs with UGG as a core motif. ESRP binding in the exon and/or in the upstream intron drives
exon skipping, whereas ESRP binding downstream of
the exon promotes exon inclusion. Thus, the position
of ESRP UGG-rich binding motifs determines an epithelial splicing regulatory ‘RNA map’. Disruption of the
epithelial splicing programme through ESRP knockdown in human mammary epithelial cells enables these
cells to change morphology and to become more motile.
Mesenchymal markers such as vimentin, fibronectin
and N‑cadherin are induced, whereas no changes of
epithelial markers such as E‑cadherin were detected.
Importantly, ESRP genes are directly downregulated by
the EMT-inducing transcription factors SNAI1, ZEB1
and ZEB2 (REFS 105,106). In addition to ESRP1 and
ESRP2, factors belonging to the RNA binding protein
FOX1 homologue (RBFOX), CUGBP Elav-like family
(CELF), muscleblind-like protein (MBNL) and heterogeneous nuclear ribonucleoprotein (HNRNP) families
of tissue-specific factors have been identified as being
involved in the regulation of EMT-specific splicing 107.
This was done using transcriptome deep-sequencing of
a TWIST1‑induced EMT model in mammary epithelial
cells and subsequent detailed motif analysis of regions
adjacent to the splice sites in EMT-regulated alternative transcripts. The importance of this more complex
view of EMT-associated alternative splicing was shown
in dedifferentiated mammary epithelial cells, which on
knockdown of RBFOX2 shifted to a more epitheliallike morphology that was marked by reduced vimentin
expression and stress-fibre presence, and by the reapp­
earance of epithelial junctional markers such as ZO1 and
α-catenin at cell–cell contacts107. These experiments are
consistent with an earlier report that the claudin-low
subtype of breast cancer (which is enriched for markers
of EMT) has a specific alternative splicing pattern that is
associated with evolutionarily conserved binding motifs
for the RBFOX2 splicing factor 108. Together, these findings
suggest a model in which a series of splicing factors collaborate to mediate splicing through position-dependent
RNA binding as part of a functional coherent splicing
regulation network (FIG. 3). Furthermore, the findings
raise the intriguing possibility that abnormal changes in
epithelial-specific splicing can steer cancer cells towards
malignant progression through a partial EMT, without the
need for canonical transcriptional reprogramming.
The relevance of EMT-associated alternative splicing
events is supported by the low expression of ESRP1 and
ESRP2 in EMT-like cancer cell lines and by the finding that
luminal B and basal breast cancer cell lines can be distinguished using splicing patterns alone, supporting the idea
that EMT-associated alternative splicing isoforms could
be used to potentially classify human breast cancer104,107.
Translational and post-translational regulation
The post-transcriptional miRNA regulatory loops
that control the translation of SNAIL and ZEB family members are well established, but more recently it
has been shown that general translational regulatory
mechanisms also have a substantial impact on EMT.
Y‑Box binding protein 1 (YB1; also known as nucleasesensitive element-binding protein 1) is a general mRNA
binding protein that, at higher concentrations in the
cell, represses translation and facilitates the storage of
5ʹ 7‑methylguanosine cap-containing mRNAs. Increased
expression of YB1 has been associated with malignant
cancer progression (TABLE 3). In RAS-transformed breast
epithelial cells, YB1 stimulates cap-independent translation
of mRNAs through internal ribosome entry site (IRES)driven translation initiation, including those encoding the EMT-associated transcription factors SNAI1,
ZEB2, lymphoid enhancer binding factor 1 (LEF1)
and TWIST1. As such, translational regulation by YB1
will not only induce EMT in those cells but it will also
potentially enhance the metastatic capacity 109.
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Exon inclusion
+
ESRP
EBSE
Epithelium-specific splicing
Non-epithelium-specific splicing
Exon skipping
ESRP –
EBSI
– ESRP
EBSI
Epithelium-specific splicing
Non-epithelium-specific splicing
Exon inclusion
+ RBFOX2
RBSE
Mesenchyme-specific splicing
Non-mesenchyme-specific splicing
Figure 3 | EMT is controlled by differential splicing events. Trans- and cis-acting
Nature
Reviews
elements controlling epithelium- and mesenchyme-specific splicing
are
shown.| Cancer
Epithelial splicing regulatory protein 1 (ESRP1) and ESRP2 regulate epithelium-specific
splicing by recognition and binding of UGG- or GGU-rich RNA sequences that are known
as ESRP binding splicing enhancer (EBSE) and ESRP binding splicing inhibitor (EBSI).
Exon inclusion or skipping is determined by the location of the EBSE and EBSI elements
relative to the alternatively spliced exons. ESRP binding to EBSI favours exon skipping
when its binding sites are located at the 5ʹ end of, and/or within, the regulated exon
motifs, whereas ESRP proteins binding at the 3ʹ end of regulated exons enhance
exon inclusion. The splicing factor RNA binding protein FOX1 homologue 2 (RBFOX2)
was shown to control mesenchyme-specific splicing. RBFOX2 binds to the UGCAUG
RNA sequence known as RBFOX2 binding splicing enhancer (RBSE) that is found
downstream of alternatively spliced exons and that favours the inclusion of the
alternative exon in the mature mRNA. Examples for epithelium- and mesenchymespecific splicing are shown for generic genes: exons are shown as boxes, and introns are
shown as horizontal lines; alternatively spliced regions are shown in blue. EMT, epithelial
to mesenchymal transition.
Ribosomal A site
During elongation cycles of
translation each
aminoacyl-tRNA enters the
ribosome at the A site where
the match is tested for the
tRNA anti-codon with the
codon of the mRNA, and where
the amino end of its amino acid
is transferred to the carboxylic
end of the nascent chain.
In addition to transcriptional induction of EMT‑TF
and activation of the miR‑200 family, TGFβ also controls EMT through the regulation of a crucial translational checkpoint at the elongation stage of EMT
effector transcripts. Blocking of translational elongation
can be accomplished with HNRNPE1 (also known as
PCBP1), which inhibits the release of elongation factor
1α1 (EEF1A1) from the ribosomal A site. The interaction
between HNRNPE1 and EEF1A1 is stabilized by binding to the stem–loop structural 33‑nucleotide-long BAT
(TGFβ-activated translation) element that is present in
the 3ʹ UTR of regulated mRNAs. On TGFβ signalling,
the BAT–HNRNPE1–EEF1A1 complex is disrupted by
AKT2‑mediated HNRNPE1 phosphorylation. It thereby
reactivates translational elongation of target EMT transcripts, such as interleukin-like EMT inducer (ILEI) and
disabled homologue 2 (DAB2), which are both involved
in malignant cancer progression110.
Most other experimental information on posttranslational regulation of EMT is available for the
EMT‑TF SNAI1, which is known to have a fast turnover time in the cytoplasm. The importance of avoiding abnormal SNAI1 protein expression is reflected by
a multitude of fine-tuned regulatory mechanisms such
as post-translational phosphorylation, lysine oxidation
and ubiquitylation1. In general, these regulatory mechanisms can be separated into glycogen synthase kinase 3β
(GSK3β)-dependent and GSK3β‑independent mechanisms of SNAI1 regulation. GSK3β is a serine/threonine
kinase that functions as a central regulator in canonical
WNT signalling and has been found to be essential for
the maintenance of epithelial differentiation111. In the
context of SNAI1 clearance, casein kinase 1 (CK1) functions as a priming kinase; the phosphorylation of SNAI1
by CK1 is required for subsequent GSK3β phosphorylation of SNAI1 at serine/threonine residues that are then
recognized and ubiquitylated by the E3 ubiquitin ligase
βTRCP1 for degradation112–114. This mechanism is mostly
counteracted by several receptor tyrosine kinase signalling pathways that stimulate SNAI1 protein stability by
inactivating GSK3β. Likewise, inflammatory cytokine
tumour necrosis factor‑α (TNFα)-mediated SNAIL stabilization is realized through nuclear factor-κB (NF‑κB),
which drives β-casein (CSN2)-controlled disruption of
the binding of GSK3β with SNAI1 and βTRCP1 (REF. 115).
The WNT–GSK3β–βTRCP1 axis was also found to
regulate SNAI2 activity that controls cancer cell EMT
programmes and to coordinately reduce BRCA1
expression116. GSK3β‑independent ubiquitin ligases,
such as MDM2 and FBXL14, have also been reported
to target SNAI1 and SNAI2 for degradation 117–119
(TABLE 3). Interestingly, it was recently reported that the
F‑box protein partner of paired (PPA) targets SNAI1,
SNAI2, TWIST1 and ZEB2 for degradation, suggesting
that there is coordinated control of these structurally
unrelated core nuclear factors implicated in EMT120.
Conversely, SNAI1 protein is stabilized by specific
phosphorylation by the kinases p21-activated kinase 1
(PAK1) and ataxia telangiectasia mutated (ATM)121,122.
Notably, these EMT-inducing kinases are activated by
DNA damage. Alternatively, direct interaction between
LOXL2 and SNAI1 stabilizes this major EMT-TF57. In
addition, hyperglycaemic conditions driving O‑β-d-Nacetylglucosamine (O-GlcNAc) modifications of SNAI1
enhance its stability 123.
By contrast, increased expression of proteases, which
are detected in nearly every cancer type, can promote
EMT induction without directly affecting EMT-TFs but
instead by modulation of signal transduction leading to
stabilization of EMT. For example, simple treatment of
mammary epithelial cells with exogenous matrix metalloproteinase 3 (MMP3) can result in a RAC1B‑mediated
increase of reactive oxygen species, which corresponds
with increased levels of SNAI1 and mesenchymal markers124. In a similar manner, MMP13 and transmembrane
protease serine 4 (TMPRSS4) affect EMT by indirect enhancement of EMT-TF activity 125,126. Although
MMPs have a direct role in extracellular matrix (ECM)
degradation, EMT can also promote this process, as it
was shown that TWIST1 is capable of promoting the
formation of invadopodia, which are specialized membrane protrusions for ECM degradation127. Likewise,
invadopodium-mediated proteolysis takes place at the
vascular basement membranes128. Interestingly, tumourassociated macrophages physically help tumour cells
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during this process by creating a microenvironment that
is permissive for invasion through epidermal growth
factor (EGF) and macrophage colony-stimulating factor 1 (CSF1) feedback signals129. As such, the study of
the role of macrophages and, more generally, of accessory cells in driving tumour-cell motility may uncover
important underlying pathways in which crosstalk with
EMT-regulatory proteins cannot be excluded.
Several lines of evidence support the idea that active
regulation of SNAI1 subcellular localization can control
nuclear activity. Furthermore, the subcellular localization of SNAI1 has a direct influence on its turnover rate,
as nuclear SNAI1 degrades more slowly than cytosolic
SNAI1 (REF. 114). The kinases PAK1 and LATS2, which are
found to be overexpressed in different epithelial cancers,
phosphorylate SNAI1, favouring nuclear retention, thus
enhancing both stability and transcriptional activity of
SNAI1 (REFS 122,130) (TABLE 3). In addition, the zinc transporter LIV1 (also known as ZIP6), a signal transducer
and activator of transcription 3 (STAT3) target, controls
the nuclear import of the SNAI1 protein131. In this context
it is worthwhile to mention that TAZ, a transcriptional
coactivator that is regulated by the Hippo tumour suppressor pathway, is phosphorylated by LATS2, attenuating its ability to induce EMT132. This inhibition of TAZ
in epithelial cells seems to depend on its association with
the cell-polarity determinant Scribble. Loss of Scribble
activates TAZ, and this endows epithelial cells with selfrenewal capabilities and links EMT-like processes with
the induction of cancer stem cell properties133. Finally, a
plausible explanation for the apparent opposing effects
of LATS2 on EMT is dependent on the cellular localization of LATS2: SNAI1 is a nuclear target and TAZ is a
cytoplasmic target134.
Export out of the nucleus is initiated by protein
kinase D1 (PRKD1), which phosphorylates SNAI1,
enabling it to bind with the epithelial specific 14‑3‑3σ
complex that shuttles it out of the nucleus. Notably,
PRKD1 expression is attenuated in different carcinoma
types and its re‑expression in prostate cancer results
Table 3 | Molecular factors involved in EMT and cancer progression: alternative splicing and post-translational regulation
Molecules
Function and involvement in EMT
Relevance to cancer
Refs
Alternative splicing
ESRP1 and
ESPR2
mRNA splicing factor regulating the formation of epithelial
cell-specific isoforms
Reduced expression in claudin-low and metaplastic
breast cancer
104
RBFOX2
RNA binding protein that regulates alternative splicing events
Not known
107
Post-translational regulation
YB1
Translation stimulation or repression dependent on YB1/mRNA
ratio
Overexpression in breast cancer cells induces EMT
109
HNRNPE1
Blocking translational elongation of EMT trancripts by binding
to the 3ʹ UTR structural element BAT. Phosphorylated by AKT2
on TGFβ signalling, resulting in reactivated translation
Repression in breast cancer cells induces EMT
199
CK1
Serine/threonine protein kinase that primes SNAI1 for
GSK3β‑mediated degradation
Repression in cells induces EMT
112
CSN2
TNFα-mediated block of SNAI1 ubiquitylation and degradation
by inhibiting SNAI1 binding with βTRCP and GSK3β
Activation in breast cancer cells on TNFα exposure
results in strengthening of the invasion and migratory
phenotype
115
MDM2
p53 E3 ubiquitin protein ligase that degrades SNAI2
Reduced expression in non-small-cell lung cancer
results in high SNAI2 expression correlated with poor
patient survival
119
FBXL14
Substrate-recognition component of some SCF (SKP1–
CUL1–F‑box protein)-type E3 ubiquitin-protein ligase
complexes interacts with SNAI1 and promotes its
ubiquitylation and proteasome degradation
Decreased expression in hypoxic colorectal tumours
117
PAK1
Serine/threonine-protein kinase that phosphorylates SNAI1,
resulting in nuclear localization
Enhanced expression in gastric cancer, non-small-cell
lung cancer, head and neck cancer, and breast cancer
LATS2
Serine/threonine protein kinase that retains SNAI1 nuclear
localization
Enhanced expression in breast cancer
LIV1
STAT3‑induced zinc transporter controlling nuclear import of
SNAI1
Enhanced expression in oestrogen receptor-positive
breast cancer and linked with its metastasis
PRKD1
Suppresses EMT through control of SNAI1 nuclear export
Downregulated in prostate cancer, breast cancer and
gastric cancer
135
CalDAG-GEFIII
Binds ZEB1 in cytoplasm, which activates RAS and attenuates
angiogenesis
Not known
137
122,
200,201
130
131,202
CalDAG-FEFII, calcium and diacylglycerol-regulated guanine nucleotide exchange factor 1; CK1, casein kinase 1; CSN2, β-casein; EMT, epithelial to mesenchymal
transition; ESRP1, epithelial splicing regulatory protein 1; FBXL14, F-box and leucine-rich repeat protein 14; GSK3β, glycogen synthase kinase 3β; HNRNPE1,
heterogeneous nuclear ribonucleoprotein E1; LATS2, large tumour suppressor 2; LIV1, solute carrier family 39, member 6; PAK1, p21 protein-activated kinase 2;
PRKD1, protein kinase D1; RBFOX2, RNA binding protein FOX1 homologue 2; STAT3, signal transducer and activator of transctiption 3; TGFβ, transforming growth
factor-β; TNFα, tumour necrosis factor-α; UTR, untranslated region.
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in MET with clear repression of mesenchymal markers and diminished metastatic capability 135 (TABLE 3).
A tight control of subcellular localization might also be
involved in the regulation of ZEB transcription factors,
as phylogenetic domain analysis revealed the probable
presence of well-conserved nuclear import and export
signals, suggesting active regulation136. Indeed, in addition to its nuclear localization ZEB1 has been found
in the cytoplasm, where it interacts with RAS guanylreleasing protein 3 (calcium and DAG-regulated)
(RASGRP3) to modulate adhesion-induced activation of RRAS in endothelial cells137. ZEB transcription
factors can also be sumoylated, disrupting the recruitment of the co‑repressor C-terminal binding protein (CTBP). ZEB2 sumoylation has been shown
to result in attenuated transcriptional repression
of CDH1 (REF. 138).
Conclusions
The abnormal induction of EMT in cancer cells has
been linked to various acquired capabilities, such as
resistance to anoikis, oncogene-induced senescence,
cell death, chemotherapy and immunotherapy alterations in DNA repair, enhanced stem cell properties,
migration, invasion and metastasis. The prominence
of these progression-associated capabilities in a cancer
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A key report describing the capacity of the
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1.
cell will vary depending on the nature and strength of
the EMT induction. In the past few years it has become
clear that EMT reprogramming of normal and tumour
epithelial cells occurs systematically at transcriptional, post-transcriptional, translational and posttranslational levels. Many different cellular stimuli
of EMT have been uncovered, whereas we have only
started to understand the many unscheduled deregulated processes featuring proteases, nuclear epigenetic
factors, protein translation-controlling factors and
alternative splicing variants that affect EMT regulation
and that are able to drive the cell towards an EMT-like
process. So far, TGFβ-mediated EMT induction clearly
exemplifies how the action of one growth factor results
in a regulatory network connecting transcriptional,
non-coding and translational levels of EMT control.
Further understanding of how the various methods
of EMT induction and their associated sophisticated
control mechanisms are interconnected and linked to
different signal transduction pathways should help us
to better understand tumour progression. Hopefully,
better insights into these different levels of regulation of
EMT will be reached in the near future and form a basis
for innovative anti-metastasis therapeutic approaches,
as well as prognostic or diagnostic markers for different
cancer types.
12.Perez-Losada, J. et al. Zinc-finger transcription factor
Slug contributes to the function of the stem cell factor
c‑kit signaling pathway. Blood 100, 1274–1286
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Acknowledgements
The authors apologize to those colleagues whose relevant
studies were not cited here. The authors’ research is funded
by grants from the VIB; the Fonds Wetenschappelijk
Onderzoek, the geconcerteerde onderzoeksacties of Ghent
University, Belgium; the Stichting tegen Kanker; the
Association for International Cancer Research, UK; and the
EU‑FP7 framework program TuMIC 2008–201662.
Competing interests statement
The authors declare no competing financial interests.
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