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doi:10.1111/j.1745-7270.2008.00442.x |
New Insights of Epithelial-Mesenchymal Transition in Cancer
Metastasis
Yadi Wu and Binhua P. Zhou*
Departments of Pharmacology and Toxicology,
and Sealy Center for Cancer Cell Biology, The University of Texas Medical
Branch, Galveston, Texas 77555, USA
Received: May 29,
2008
Accepted: June 9,
2008
This work was
supported by grants from the John Sealy Memorial Endowment Fund, a pilot award
from the ACS (IRG-110376), the Susan G Komen Foundation (KG081310) and NIH (RO1CA125454) (to B.P. Zhou), and the post-doctoral fellowships from NIH (T32CA117834) (to Y. Wu)
*Corresponding
author: Tel, 409-747-1963; E-mail, [email protected]
Epithelial-mesenchymal transition (EMT) is a key step during
embryonic morphogenesis, heart development, chronic degenerative fibrosis, and
cancer metastasis. Several distinct traits have been conveyed by EMT, including
cell motility, invasiveness, resistance to apoptosis, and some properties of
stem cells. Many signal pathways have contributed to the induction of EMT, such
as transforming growth factor-b, Wnt, Hedgehog, Notch, and nuclear factor-kB. Over the last
few years, increasing evidence has shown that EMT plays an essential role in
tumor progression and metastasis. Understanding the molecular mechanism of EMT
has a great effect in unraveling the metastatic cascade and may lead to novel
interventions for metastatic disease.
Keywords epithelial-mesenchymal transition; metastasis; Snail; Twist;
signal transduction
Although 90% of cancer deaths are caused by metastasis, the
pathogenesis and mechanism underlying this event remains poorly defined.
Understanding this process will provide great promise for the discovery of
novel therapeutics for treating metastatic cancer. Metastasis is a ‘hidden’
event, which happens inside the body and is difficult to examine. It is
believed to consist of four distinct steps: invasion, intravasation,
extravasation, and metastatic colonization [1,2]. During invasion, tumor cells
lose cell-cell adhesion, gain mobility, and leave the site of the primary tumor
to invade adjacent tissues. In intravasation, tumor cells penetrate through the
endothelial barrier and enter the systemic circulation. In extravasation, cells
that survive the anchorage-independent growth conditions in the bloodstream
attach to vessels at distant sites and leave the bloodstream. Finally, in
metastatic colonization, tumor cells form macrometastases in the new host
environment [1,2]. Using in vivo video microscopy and quantitative
approaches, the first step, the acquisition of invasive ability and motility,
is found to be the rate-limiting step in the metastatic cascade [1,3]. Beyond
this step, survival of tumor cells in the circulation, their arrest in a
distant organ, and their initial extravasation are relatively efficient
processes. These findings clearly indicate that understanding the initial step
of metastasis is critical to the future development of novel strategies to
prevent cancer metastasis. Epithelial-mesenchymal transition (EMT), a process
vital for morphogenesis during embryonic development, is attracting increasing
attention as an important mechanism for the initial step of metastasis. Here we
highlight the significance of EMT in cancer development and our emerging
understanding of its regulation in tumor metastasis. We present some of the
current mechanisms parallel between their known roles in EMT induction during
development and how these processes can be hijacked by tumor cells to enhance
metastasis.
EMT is a Critical Cellular Process
EMT, a process vital for morphogenesis during embryonic development,
was first recognized as a feature of embryogenesis in the early 1980s [4,5].
During gastrulation in Drosophila flies and mammals, cells migrate from
an epithelial-like structure to spatially reorganize one of the three main
embryonic layers, the mesoderm [4,5]. In this EMT process, epithelial cells
acquire fibroblast-like properties and show reduced intercellular adhesion and
increased motility [4,6]. EMT is essential for many morphogenetic events, such
as gastrulation and organogenesis in embryonic development, tissue remodeling,
fibrosis and wound healing, and heart development [7,10]. The migratory nature
of these cells has prompted comparisons with metastatic cells and attracts
increasing attention as an important mechanism for the initial step of
metastasis, since genes implicated in EMT during embryogenesis have been shown
to control metastasis [5,6]. Some pathologists were initially skeptical of this
theory because they could not conclusively determine that EMT was apparent in
human tumor specimens [11]. However, a growing body of evidence strongly
suggests that EMT is a critical early event for the invasion and metastasis of
many carcinomas [12,13]. A hallmark of EMT is the loss of E-cadherin
expression, an important caretaker of the epithelial phenotype [4,14].
E-cadherin is a cell-cell adhesion molecule that participates in homotypic,
calcium-dependent interactions to form epithelial adherent junctions [15,16].
Loss of E-cadherin expression is consistently observed at sites of EMT during
development and cancer, and the E-cadherin expression level is often inversely
correlated with the tumor grade and stage [15,16]. Numerous studies have shown
that virtually all cases of invasive lobular carcinoma, which accounts for 8%
of all breast cancers, have loss of E-cadherin expression as a result of
E-cadherin gene mutation and promoter hypermethylation [17,18]. However,
patients with invasive ductal carcinoma (IDC), which accounts for 80% of all
breast cancers, retain E-cadherin expression. Thus, dominant transcriptional
repression is mainly responsible for the transient loss of E-cadherin
expression during the metastatic progression of IDC [19,23].
Several transcription factors have been implicated in the
transcriptional repression of E-cadherin, including zinc finger proteins of the
Snail/Slug family, Twist [14,24–27], dEF1/ZEB1, SIP1, and the basic helix-loop-helix factor E12/E47 [28–34]. These repressors
can also act as molecular triggers of the EMT program by repressing a subset of
common gene that encode cadherins, claudins, cytokines, integrins, mucins,
plakophilin, occluding, and zonula occludens (ZO) proteins to promote EMT [10].
Strikingly, all of these transcriptional repressors are best known for their
roles in early embryogenesis. The first discovered and most important of these
repressors is Snail, a DNA-binding factor that was identified in Drosophila
as a suppressor of the transcription of shotgun (an E-cadherin homolog)
in the control of embryogenesis [35,36]. Snail has a central role in
morphogenesis, as it is essential for the formation of the mesoderm and neural
crest, which requires large-scale cell movements in organisms ranging from
flies to mammals. Absence of Snail is lethal because of severe defects at the
gastrula stage during development [37]. Expression of Snail represses
expression of E-cadherin and induces EMT in MDCK (Madin-Darby Canine Kidney)
and breast cancer cells [38–40], indicating that Snail plays a fundamental role in EMT and
breast cancer metastasis by suppressing expression of E-cadherin. Microarray
analyses of primary human breast cancers suggest that a high level of Snail
expression is correlated with a poor clinical outcome in women with early-stage
breast cancer [41,42]. In fact, overexpression of Snail was recently
found in both epithelial and endothelial cells of invasive breast cancer but
was undetectable in normal breast [43,44]. Some studies indicated that Snail
was implicated in the initial migratory phenotype of primary tumors and
considered as an early marker of EMT. In contrast, Slug, ZEB1, ZEB2/SIP1, and
Twist could be responsible for the maintenance of migratory cell behavior,
malignancy and other tumorigenic properties. However, this model awaits more
detailed analysis owing to specific and independent roles of the different
factors.
Microenvironment Signals, Developmental Pathways, and EMT
EMT is a dynamic process and is triggered by stimuli that emanate
from microenvironments, including extracellular matrix (such as collagen and
hyaluronic acid) and many secreted soluble factors, such as Wnt, transforming
growth factor-b (TGF-b), Hedgehog, epidermal growth factor (EGF), hepatocyte growth factor
(HGF), and cytokines [45]. The major task is to delineate the signaling
pathways mediated by these microenvironmental stimuli in initiating and
controlling EMT and cancer metastasis. Among many of these signaling pathways,
Wnt, TGF-b, Hedgehog, Notch, and nuclear factor-kB (NF-kB) signaling
pathways are found to be critical for EMT induction. These signaling pathways
orchestrate a concerted and elaborate gene program and protein network needed
for the establishment of mesenchymal phenotypes after disassembly of the main
elements of epithelial architecture, such as cell-cell junctions and cell
polarity. As many of these normal developmental pathways are also involved in
EMT, morphogenesis, and motility during development, it is not surprising that
tumor cells usurp these pathways for their own purposes.
The Wnt/b-catenin pathway has a particularly tight link with EMT [46]. On one
hand, b-catenin is an essential component of adherent junctions, where it
provides the link between E-cadherin and a-catenin and modulates
cell-cell adhesion and cell migration. On the other hand, b-catenin also
functions as a transcription cofactor with T cell factor (TCF). In unstimulated
cells, the level of free cytoplasmic b-catenin is kept low through a destruction
complex, which consists of axin, adenomatous polyposis coli (APC), GSK-3b, and casein
kinase (CKI). GSK-3b phosphorylates b-catenin and triggers its ubiquitination and degradation by b-Trcp. In the
presence of Wnt ligands, Wnts bind to frizzled and LRP5/6 receptor complexes to
inactivate GSK-3b in the destruction complex. This, in turn, results in the
stabilization and nuclear accumulation of b-catenin and leads to the
transcription of Wnt target genes, such as c-myc, cyclin D, and survivin [47].
Nuclear translocation of b-catenin can activate the expression of Slug and thus induces EMT.
Expression of b-catenin in oocyte induces a premature EMT in the epiblast,
concomitant with Snail transcription. Interestingly, Snail is a highly unstable
protein and is dually regulated by protein stability and cellular location. We
showed that GSK-3b binds and phosphorylates Snail at two consensus motifs to dually
regulate the function of this protein: phosphorylation at the first motif
regulates its ubiquitination mediated by b-Trcp, whereas
phosphorylation at the second motif controls its subcellular localization [40].
Thus, Wnt can suppress the activity of GSK-3b and stabilizes the protein
level of Snail to induce EMT and cancer metastasis [48,49]. Whether the
synergistic activation of Snail and b-catenin by Wnt signaling pathway is required
for EMT induction and metastasis of tumor cells remains to be defined.
TGF-b is a potent inducer of EMT. It not only contributes to EMT during
embryonic development but also induces EMT during tumor progression in vivo [50].
Overexpression of Smad2 and Smad3 results in increased EMT in a mammary
epithelial model [51]. Knockout of Smad3 blocks TGF-b-induced EMT in primary
tubular epithelial cells, and the reduction of Smad2 and Smad3 function is
associated with the decreased metastatic potential of breast cancer cell lines
in a xenograft model [52]. TGF-b can also downregulate various epithelial proteins, including
E-cadherin, tight junction protein ZO-1, and several specific keratins, and
also upregulates certain mesenchymal proteins such as fibronectin,
fibroblast-specific protein 1, a-smooth muscle actin, and vimentin. In addition, TGF-b cooperates with
numerous kinases such as RAS, MAPK, p38MAP to promote EMT [50,53]. Furthermore,
TGF-b cross-talks with other signal pathways and coordinates the
regulation of EMT. Recent reports suggest that functional interactions between
TGF-b with Notch, Wnt, and NF-kB contribute significantly to the induction of
EMT [50].
The Hh signaling pathway was first identified in a large screen for Drosophila
genes required for patterning of the early embryo [54,55]. Analysis of the Hh
mutant, named after its prominent phenotype (epidermal spikes in larval
segments that normally are devoid of these extensions) led to the cloning of
the Hh gene. The Hh ligands, Sonic (Shh), Desert (Dhh), and Indian (Ihh) in
vertebrates and Hh in Drosophila, are secreted proteins that undergo
several posttranslational modifications to gain full activity. Key effectors of
Hh signaling include zinc-finger proteins of the Gli1-3 transcription
factors. Hh signaling can initiate cell
growth, cell division, lineage specification, and axon guidance and can also
function as a survival factor. In light of this range of biologic functions, it
is not surprising that mutations in components of the Hh pathway are associated
with both embryonic developmental defects and tumor progression. Indeed,
mutations in Patched (PTC) and/or Smoothened (SMO) trigger inappropriate
activation of the Hh pathway and have been identified in basal cell carcinoma,
rhabdomyosarcoma, medulloblastoma, and other tumor types [54,55]. In mouse
epidermal cells or in rat kidney epithelial cells immortalized with adenovirus
E1A, Gli1 rapidly induces transcription of Snail and promotes EMT [56,57].
Targeted expression of Gli1 in the epithelial cells of mammary gland of mice
induces the expression of Snail and thus results in the disruption of the
mammary epithelial network and alveologenesis during pregnancy [58].
Conversely, blockade of Hedgehog signaling by inhibitor cyclopamine suppresses
pancreatic cancer invasion and metastasis through inhibiting EMT in the
pancreatic cancer cells [59].
Notch is an evolutionarily conserved signaling pathway that
regulates cell fate specification, self-renewal, and differentiation in
embryonic and postnatal tissues. Four Notch (Notch 1–4) and five ligands
(Jagged1, 2 and Delta-like1, 3, 4) have been identified. Notch signaling is
normally activated followed by ligand-receptor binding between two neighboring
cells, Notch undergoes intramembrane cleavage by g-secretase and its
intracellular domain (NICD) is released and translocates to the nucleus to
activate gene transcription by associating with Mastermind-like 1 (MAM) and
histone acetyltransferase p300/CBP. Alteration of Notch signaling has been
associated with various types of cancer in which Notch may act as an oncogene or
as a tumor suppressor. The observation that Notch pathway is required for EMT
was first made during cardiac valve and cushion formation at heart development
[60]. This implies that Notch acting through a similar mechanism, may also be
involved in the EMT induction during tumor progression and converts polarized
epithelial cells into motile, invasive cells [61]. Indeed, overexpression of
Jagged1 and Notch1 induces the expression of Slug and correlates with poor
prognosis in various human cancers [62]. Slug is essential for Notch-mediated
EMT by repressing E-cadherin expression, which results in b-catenin
activation and resistance to anoikis. Inhibition of Notch signaling in
xenografted Slug-positive/E-cadherin-negative breast tumors promotes apoptosis
and inhibits tumor growth and metastasis [62]. In addition, Notch signaling
deploys two distinct mechanisms that act in synergy to control the expression
of Snail [63]. First, Notch directly upregulates Snail expression by
recruitment of the Notch intracellular domain to the Snail promoter, and
second, Notch potentiates hypoxia-inducible factor 1a (HIF-1a) recruitment to
the lysyl oxidase (LOX) promoter and elevates the hypoxia-induced upregulation
of LOX, which stabilizes the Snail protein. Thus, Notch signaling is required
to convert the hypoxic stimulus into EMT, increased motility, and invasiveness
of tumor cells.
NF-kB is another key modulator for EMT. Recently, NF-kB was identified
as a central mediator of EMT in a model of breast cancer progression [6,64]. In
this model, the NF-kB signaling pathway was essential for distinct aspects of EMT
(apoptosis protection, EMT induction and maintenance) as well as being required
for metastasis. This suggests that both Ras- and TGF-b-dependent effects on EMT,
including activation of many EMT-specific genes, are mediated, at least in
part, via NF-kB activity [6]. Interestingly, the E-cadherin repressors Twist and
Snail have been suggested as possible downstream targets of NF-kB [6,65,66].
Cell Polarity and EMT
During EMT, epithelial cells lose cell-cell junctions and polarity,
leading to a more migratory, fibroblast-like “mesenchmymal” cell phenotype. Many
studies have emphasized the major role of signaling pathways leading to the
transcriptional repression of the E-cadherin in adherens junction by Snail,
Slug, SIP1, and Twist. Little is known about how EMT disrupts the formation of
tight junction and cell polarity. Polarity is largely regulated by a conserved
set of proteins known as partition-defective (PAR) proteins, which are required
for organizing the basal-apical polarity of
epithelial cells and for the establishment and maintenance of apical junction.
The PAR3/PAR6/aPKC complex localizes selectively at the apical junction and the
apical plasma membrane; whereas Par1, resides at the basolateral membranes of
epithelia. Mutual antagonistic interactions between these two complexes results
in the formation of cellular and functional asymmetry within the cell. In
addition to the Par complex, the lateral resided CRUMBS/PALS1/PATJ complex and
the tight junction associated SCRIBBLE/DLG/LGL complex, are also required for
the formation of cell polarity. During the initial stage of epithelial cell
contact, spot-like adherens junctions first appear at the tips of protrusions that
contain E-cadherin, nectins, junctional adhesion molecule (JAM), and protein
ZO-1. E-cadherin mediates initial intercellular adhesion, which is
substantially strengthened after its connection to the actin cytoskeleton
through a– and b-catenin. These connections mature into adherent junctions and
promote the formation of tight junctions, which further anchors to the Par
complex to establish cell polarity. Recent work has shown that TGF-b can induce
phosphorylation of Par6, which in turn stimulates binding of
Par6 to E3 ligase Smurf1. The Par6-Smurf1 complex then mediates the localized ubiquitination of RhoA to dissolute tight junctions
during EMT [67]. TGF-b can also downregulate the Par3 expression to destroy the cell
polarity [68]. Whiteman et al also showed that Snail disrupted the
apical polarity complex by inhibiting the expression of Crumb3 [69]. In
addition, ZEB1 suppresses the expression of Lgl2, Crumbs3, HUGL2 and PATJ to
disrupt cell polarity [70,71]. Thus, it becomes obvious that the disruption of
tight junctions and cell polarity represents a new trait of EMT.
EMT in Cell Survival and Tumor Recurrence
During EMT, epithelial cells detach from the extracellular matrix
(ECM), which triggers the apoptotic process. The ability to survive in the
absence of normal matrix components represents an important property for cells
undergoing EMT. Several known apoptotic and anti-apoptotic proteins are
involved in EMT. Overexpression of Bcl-2 and Bcl-XL increases the metastasis
capacity of epithelial cells without affecting primary-tumor formation [72,73].
Moreover, integrin-mediated signaling is also attributable to the inhibition of
cell death. For example, focal-adhesion kinase (FAK), a crucial activator of
the tyrosine-kinase pathway, is associated with the intracellular tails of
integrin and its activation is sufficient for epithelial cell survival [74]. In
mouse embryo, FoxD3 requires the concomitant expression of SOX9 and Slug to induce
EMT. Sox9 can inhibit cell death and specifies the neural-crest cell lineages
[75].
Snail and Slug act as inhibitors of apoptosis through several
mechanisms. Slug negatively regulates the expression of the pro-apoptotic p53
and Puma [76–78], while Snail represses Cyclin D2 transcription and
increases the p21Cip1/Waf1 level and concomitantly activates the
MAPK and PI3K survival pathway to confer resistance to the lethal effects of serum depletion or TNFa administration [79–81]. Similarly,
Twist, recently involved in breast cancer metastasis through regulation of EMT,
functions as an oncogene in many human cancers. Twist also negatively regulates
apoptosis during both embryogenesis and tumor progression [82]. All of these
transcription factors exert the role in cell survival, differentiation and
metastasis. Thus, increased expression of these transcription factors during
EMT is sufficient to overcome cell death provoked by proapoptotic signals. They
provide a selective advantage for the invasive cells to migrate through hostile
territories. This anti-apoptotic
function is essential for the migratory cells to reach their final destinations
during embryogenesis and is also important for malignant cells to disseminate and
form metastases.
Using a mammary-specific, inducible
HER2/Neu transgenic mouse model, Moody et
al demonstrated that EMT occurred in tumor recurrence and Snail was
upregulated spontaneously [42]. Snail is sufficient to induce EMT in
HER2/Neu-induced primary tumor cells and to promote rapid tumor recurrence in
vivo following downregulation of the HER2/Neu pathway. Consistent with
this, breast cancer relapses in Wnt1 transgenic mice lacking either Ink4a/Arf
or p53, and this relapse is accompanied with EMT with robust Snail expression
and undetectable E-cadherin [83]. Moreover, ZEB1 is an important transcription
factor that regulates EMT. It also maintains the proliferation of a subset of
progenitor cells in gestation. The proliferative defects occur in the ZEB1
mutant mice and lead to premature replicative senescence in cultured MEFs. This
cellular senescence is triggered by two cell cycle inhibitors, p15Ink4b and p21Cip1/Waf1 [84]. Together, EMT may foster oncogene-independent
survival of a crucial subset of tumor cells to promote tumor progression.
EMT and Cancer Stem Cell
In addition to the gain of anti-apoptotic ability for cells
undergoing EMT, Weinberg’s group recently demonstrated that EMT also generates
properties of stem cells, such as self-renewal [85,86]. Ectopic expression of
Snail or Twist yields great increases in their ability to form mammosphere,
which represents the presence of epithelial stem cells. Similarly, EMT
generates more mammary epithelial stem-like cells from more differentiated
populations of normal mammary epithelium. Surprisingly, the stem-like CD44high/CD24low cells exhibit strong reduction of
E-cadherin, significant increased expression of fibronectin and vimentin, and
robust levels of FOXC2, Snail, Twist and Slug. Consistent with this finding in
mammary epithelial cells, the differentiation of human embryonic stem (ES)
cells is also associated with all the characteristic EMT events, including
repression of E-cadherin, increasing expression of vimentin, upregulation of
Snail and Slug, high activity of gelatinase, and enhanced cell motility [87].
EMT seems to be the definitive step in human ES differentiation. Thus, EMT
enables cancer cells not only to disseminate from a primary tumor but also to
form the macroscopic metastases with self-renewal capability.
EMT and microRNA
As EMT plays a central role in
embryogenesis, fibrosis, wound healing, and cancer metastasis, it is not
surprising that a bewildering number of regulators associate with this
fundamental process. Recently, microRNA has appeared as a powerful master
regulator of EMT. MicroRNAs are small 20–22-nucleotide long noncoding RNAs that
modulate gene expression at the post-transcriptional level [88,89]. MicroRNAs
have been implicated in regulating diverse cellular pathways, such as cell
differentiation, proliferation and programmed cell death and are commonly
dysregulated in human cancer. Recent findings suggest that microRNAs also
contribute to EMT. For example, Twist induces microRNA-10b transcription, which
inhibits the translation of HOXD10 and results in elevated expression of RhoC,
and thus facilitates cancer cell metastasis. Significantly, the level of
miR-10b expression in primary breast carcinomas correlates with clinical
progression [90]. In addition, several reports demonstrated that the
microRNA-200 family were markedly
downregulated in cells that had undergone EMT in response to TGF-b [91–94]. Because
microRNA-200 directly targets the mRNA of ZEB1 and SIP1, expression of miR-200
induces upregulation of E-cadherin in cancer cell lines and suppresses their
motility. Consistent with their role in regulating EMT, loss of microRNA-200 is
commonly found in invasive breast cancer cell lines with mesenchymal phenotype
and in regions of metaplastic breast cancer specimens lacking E-cadherin. In
addition, an EMT specific microRNA miR-21 is found in TGF-b-induced EMT in
human keratinocytes, a model of epithelial cell plasticity for epidermal injury
and skin carcinogenesis [95]. MiR-21 is abundantly expressed and associated
with carcinogenesis. It targeted two tumor suppressors, tropomyosin 1
(TPM1) and programmed cell death-4 (PDCD4) to modulate the cell
proliferation, microfilament organization, and anchorage-independent
growth [96,97]. Interestingly, microRNA can target distinct functions in
different signaling pathways and thus contributes to several key events
associated with tumor progression. Therefore, targeting microRNA can be a good
therapeutic approach for cancer prevention and treatment with the effect of
“one stone hitting multiple birds”.
Future Perspectives
During the past few years, EMT has emerged as one of the hottest
medical science topics. The role of EMT in tumor progression and metastasis
provides an intriguing mechanism to explain the initial step of metastasis.
However, several areas are required for further investigation to
comprehensively understand the role of EMT in physiological and pathological
processes. First, most traditional EMT markers are found in scenarios other
than EMT. New markers of EMT are required to better distinguish EMT. Second,
EMT is a kinetic conversion that varies considerably from hours to weeks. Other
cellular evens might embed in the EMT program. It is difficult and challenging
to obtain informative results on gene expression and to discriminate between
general and cell/stag-specific molecular players that are responsible for EMT.
Third, additional studies are required to understand the molecular mechanisms
controlling EMT. The crosstalk between different signal pathways and molecules
is a crucial issue to elucidate the complicated regulation of EMT, such as the
communications between cadherin and integrin, Snail and b-catenin, TGF-b PDGF. Finally,
better models are particularly required to study EMT in vivo and
powerful imaging is also needed to unveil the behavior of migratory cells in
real time. New discoveries will elucidate the complex strategies of EMT and
hold great promise for yielding novel therapeutic approaches for treating
cancer.
Acknowledgements
We apologize to the many contributors whose work in this field is
important but we were unable to cite here.
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