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10.1182/blood-2003-08-2807
ENVIRONMENTAL GUIDANCE OF NORMAL AND TUMOR CELL PLASTICITY:
EPITHELIAL MESENCHYMAL TRANSITIONS AS A PARADIGM
Gregor Prindull and Dov Zipori
Department of Molecular Cell Biology, the Weizmann Institute of Science, Rehovot, 76100,
Israel
Short title for running head: Environmental control of plastic cell transitions
Word count: 5532
Corresponding author: Dov Zipori, Ph.D.
Dept. of Molecular Cell Biology,
Weizmann Institute of Science,
Rehovot, 76100, Israel
Tel: 972-8-9342484
Fax: 972-8-9344125
E-mail: [email protected]
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Copyright (c) 2004 American Society of Hematology
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Abstract
Epithelial mesenchymal transitions are a remarkable example of cellular plasticity. These
transitions are the hallmark of embryo development, are pivotal in cancer progression, and
seem to occur infrequently in adult organisms. The reduced incidence of transitions in the
adult could result from restrictive functions of the microenvironment that stabilizes adult cell
phenotypes and prevents plastic behaviour. Multipotential progenitor cells exhibiting a
mesenchymal phenotype have been derived from various adult tissues. The ability of these
cells to differentiate into all germ layer cell types, raises the question as to whether
mesenchymal epithelial transitions occur in the adult organism more frequently than presently
appreciated. A series of cytokines are known to promote the transitions between epithelium
and mesenchyme. Moreover, several transcription factors and other intracellular regulator
molecules have been conclusively shown to mediate these transitions. However, the exact
molecular basis of these transitions is yet to be resolved. The identification of the restrictive
mechanisms that prevent cellular transitions in adult organisms, that seem to be unleashed in
cancerous tissues, may lead to the development of tools for therapeutic tissue repair and
effective tumor suppression.
Introduction
Cell fate is determined by a variety of factors that control gene transcription/silencing during
embryonic development and in normal adult physiology. Traditionally, cell commitment to
differentiation has been viewed as consisting of a series of irreversible steps. A descending
hierarchy of diminishing capacities of differentiation is began with the totipotent embryonic
stem cell, capable of giving rise to all cells of the embryo. Embryonic stem cells (ES) that
originate from the inner cell mass (ICM) were considered pluripotent. In the first step of
differentiation, at compaction, embryonic cells loose their ability to differentiate into cells of
the placenta but maintain their capacity to differentiate into any cell of the embryonic
organism plus a few extra-embryonic cell types. During subsequent stages of organogenesis,
the potency of differentiation is further reduced, coupled with a diminished proliferative
capacity. Recent experiments have shown that this traditional one-way street of progressive
commitment to differentiation may not be irreversible. A recent speculative hypothesis for
hematopoiesis 1 suggests that stem cell systems may not be hierarchical but rather show
considerable degrees of plasticity. Changes in functional stages may depend on cell cycle
transit with gene expression to vary widely, depending on shifting chromatin constellations.
Stem cells appear to continuously change surface receptor expression 2 and thus respond
differently to external stimuli at different points of the cell cycle. Nuclear transfer
experiments have shown that fully differentiated nuclei of mature adult cells are converted
into ES-like nuclei, with corresponding pluripotency, by cytoplasmic factors of an oocyte 3,4.
Furthermore, as suggested by a multitude of recent publications, adult stem cells are by far
more plastic than previously thought (reviewed in 5) and may undergo transdifferentiation.
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Figure 1: The alleged plasticity of adult stem cells: hemopoietic stem cells (HSC) which are well known for their
ability to give rise to all blood cells have been now reported to give rise to cells derived from the three
embryonic germ layers. The question mark relates to the opposing reports related to this issue. Whereas
mesenchymal stem cells (MSC) were first shown to give rise to mesodermal derivatives only, one sub-type of
these adult stem cells, multipotential adult progenitor cells (MAPC), apparently differentiate into a wide range of
cell types of all germ layers.
To indicate just a few examples, neural stem cells generate blood 6 and skeletal muscle 7, and
contribute to several embryonic tissues upon implantation into blastocysts 8. Hematopoietic
stem cells (HSC) give rise to epithelium 9 and mesangial kidney cells 10, while multipotent
adult stem cells from the dermis give rise to neurons, glia, smooth muscle, and adipocytes 11,
and bone marrow derived cells turn into hepatocytes 12 (Figure 1). Some of these reports, lack
a final proof that transition among lineages has, in fact, occurred at a single cell level. It has
not been excluded, beyond reasonable doubt, that stem cells of various lineages are present
within the same tissue. Indeed, the muscle seems to contain a variety of cell types including a
population of muscle specific stem cells, and other stem cells that have hemopoietic potency,
that seem to be of bone marrow origin and do not have significant myogenic activity 13.
However, an additional recent study suggests that muscle derived stem cells (MDSC)
transdifferentiate into hemopoietic stem cells but nonetheless retain their myogenic potential
14
. The study of Krause et al. 9 demonstrated the transition of rare bone marrow seeking HSC
into epithelium, but other investigators were unable to repeat these results and suggested that
HSC are not capable of significant non-hematopoietic transdifferentiation 15. In addition,
recent studies show that the ability of bone marrow derived cells to turn into hepatocytes, that
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was interpreted as representing a process of transdifferentiation, is in fact due to cell fusion
16,17
. These discrepancies should be resolved by further experiments. Yet, stem cells, other
than HSC, are found in the bone marrow. One type is the mesenchymal stem cell (MSC),
known for its ability to differentiate into mesoderm derived tissues such as muscle,
cardiomyocytes, bone, cartilage and fat 18. Recent studies indicated that a population of such
stem cells, termed multilineage adult progenitor cells (MAPC), may be derived from the bone
marrow and other sites, in both mouse and man 19 (Figure 1). Single MAPC clones
transdifferentiate into ectoderm and endoderm derived tissues, produce blood cells, and
repopulate embryonic tissues in vivo upon inoculation into a blastocyst. This phenomenon of
transdifferentiation occurs in cells that have been removed from their natural habitat and
subjected to a specific set of conditions ex vivo, prior to their reimplantation. This may mean
that, in situ, MAPCs, or their precursors, seldom exhibit their full potential plasticity. This
point would be testable only once the precursors of MAPCs have been identified in situ. In
any event, the study strongly suggests that the microenvironment is suppressing stem cell
plastic behaviour: It is their displacement into culture that causes MAPC accumulation
(following 30-40 population doublings) and reintroduction into adult or embryonic
environments promotes differentiation. Multicellular tissue environments seem therefore to
impose restrictions on cell phenotypes. Such restrictions have been suggested to participate in
creating the hematopoietic microenvironment of blood forming tissues 20. This model
suggests that stromal cells impose restrictions, that prevent invasion and accumulation, or
block differentiation of cells, which otherwise could disrupt tissue integrity.
This review is not intended to resolve the heated debate over the extent of plasticity of stem
cells 21. Rather, we aim to discuss, in detail, one aspect of plastic cell behaviour, i.e. the
process of epithelial-mesenchymal transition (EMT), which is a hallmark of development.
EMT, and the reciprocal phenomenon of mesenchymal-epithelial transition (MET), both
entail drastic phenotypic and functional changes. Thus, an immobile epithelial cell, bound by
cadherin bridges to adjacent cells, becomes a mobile, fibroblast-like mesenchymal cell that
looses its cell-cell contacts, in EMT, while the reverse occurs during MET (Figure 2). In both
EMT and MET, plasticity is indisputable in cases wherein these processes are observed at a
single cell level. We shall highlight the dominant role of the microenvironment in the
regulation of these processes and argue that in the adult organism there are tighter restrictions
on such plasticity, than during embryonic development, making it a less frequent
phenomenon. Nevertheless, phenotypic plasticity, in regard to the capacity to switch lineage
and direction of differentiation, is apparently an inherent property of cells, which they never
totally loose. It is the task of future research to discover the molecular basis of the restrictive
mechanisms that attenuate the plastic behaviour of cells, so that plasticity could be harnessed
effectively for medical uses.
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Figure 2: EMT entails loss of cell junctions, detachment form fixed ECM adherence and further induction of
motility, whereas MET is the reverse phenomenon. These processes may occur sequentially during
embryogenesis.
Epithelial Mesenchymal Transditions at the Cellular Level
EMT profoundly affects major cell properties. This includes transcriptional downregulation of
epithelial cell markers and up-regulation of mesenchymal markers including cell motility and
mesenchymal molecules such as fibronectin, stromelysin-1 (a matrix metallo-proteinase) 22,
collagen I, vimentin and tenascin. Vimentin is a marker of highly motile stromal fibroblasts,
and of late stage metastatic progression 23-25. Additional mesenchymal criteria of embryonic
cell plasticity include reorganization of the cytoskeleton and degradation of basal lamina.
Conversely, in MET, epithelial cell markers including E-cadherin, a homophilic cell-cell
adhesion molecule, are up-regulated, and the cytoskeleton with fibroblast-like actin fibers
reorganizes. EMT occurs in diverse steps of normal embryonic development, as well as in
carcinogenesis (see below). During embryonic development, epithelial-mesenchymal
interactions are intimate. EMT is part of the formation of the extra-embryonic parietal
endoderm, the ICM, branching morphogenesis of lungs, kidneys and mammary gland
epithelium. Since, initially, the ICM consists exclusively of epithelial cells, generation of the
primary ICM mesenchyme must be initiated, in a subpopulation of these cells, by alterations
of gene expression. These changes consist in changes in adhesion bindings of epithelial cells
to the extracellular matrix (ECM) and to neighbouring cells, and in the interruption of direct
intercellular information by an exchange of trafficking molecules through gap junctions 26-28
(reviewed in 29,30). Embryonic cells in transition to mesenchymal cells develop cytoplasmic
actin based machinery for invasion of, and migration throughout, the ECM. During migration,
they receive specific, variable information from the ECM that, in turn, they modify by
secreting factors.
The primary mesenchyme constitutes the earliest, motile form of the ICM stroma 31. It has
been suggested that in the course of further embryonic development, “oscillations” occur
between EMT and MET, i.e. between the “stable” epithelia and an “unstable” mesenchyme
with plastic and exploratory capacities 32 (Figure 2). In fact, EMT and MET appear to be
reversible processes 33, even in neoplastic cell growth 34. Primary mesenchyme appears to be
converted into secondary epithelia which, in turn, give rise to secondary mesenchyme and
tertiary epithelial structures, e.g. in the urogenital tract. The ability to go through cycles of
epithelial and mesenchymal states makes the mesoderm highly plastic and versatile,
properties that are major contributors of development of complex organisms 32 (Figure 2). An
embryonic carcinoma model of EMT indicates that this process may be steered by
extracellular factors including parathyroid hormone-related peptides 35. All vertebrates
employ MET in embryonic somitogenesis for the generation of segmental plates. The best
known example is the conversion of metanephrogenic tissue into excretory tubular epithelium
in kidney development, but it also takes place in the ontogeny of the gastro-intestinal tract, the
lungs, and skin 9. In the placenta, the extravillous cytotrophoblast undergoes EMT losing
some of its epithelial features (E-cadherin, integrin α6β4) but retaining others (e.g.
cytokeratins) (reviewed in 36). Mesenchymal cells that do not regain an epithelial state
through MET, differentiate and give rise to muscle, bone, nerve, or connective tissue
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(reviewed in 32), and form the stromal microenvironment of the hemopoietic organs, and other
tissues.
Plastic transitions between cell types, which are fundamental in development, may also occur
in the adult organism. It has been suggested that EMT is a feature of pathologic disease states.
In biopsy material, progressive stages of EMT have been described in different renal diseases
37
. In chronic renal interstitial fibrosis, transforming growth factor (TGF) β1 induced EMT
with increased matrix mobility and invasive capacities 38. In adult organisms, processes of
angiogenesis entail many traits of cellular transitions and are similarly regulated by common
cytokines (reviewed in 32). A recent study, aimed at determining whether tissue repair
involves lineage switches, shows that single spinal cord cells turned into muscle or cartilage
during amphibian tail regeneration 39. In view of the proposed plasticity of adult mesenchymal
stem cells, one should consider the possibility that tissue damage in the adult, such as
following wounding, is followed by differentiation and transdifferentiation of mesenchymal
stem cells. We propose to re-visit wound healing and other regenerative processes, bearing in
mind the possible contribution of the mesenchyme to epithelial organization by MET.
The dramatic changes in cell phenotypes, that occur during embryogenesis, and possibly also
in the adult organism, in the course of EMT and MET, are induced by environmental factors,
the nature of which is discussed in the next section.
The Molecular Basis of Epithelial Mesenchymal Transitions
Communication among neighbouring cells, and between cells and their microenvironment,
are fundamental requirements for a synchronized development and correct differentiation. The
microenvironment is the major driving force in embryogenesis, already in the ICM. Functional coordination, timing and synchronization, and spatial segregation of embryonic cells
are mediated by the ECM, cytokines 40,41, adhesion molecules, membrane receptors, and
intercellular junctions (reviewed in 30,42). Rearrangement of the ECM occurs by necessity in
EMT and MET. The role of the microenvironment in determining gene transcription has been
dramatically demonstrated by injection of differentiated somatic epidermal cells 43,44 and
human adult multipotent stem cells 19 into blastocysts. The injected cells acquired lineage
differentiation characteristics in accordance with the respective organs in the recipient
embryo.
As mentioned above, E-cadherin contacts between epithelial cells are lost during EMT. Ecadherin junctional adhesion complexes form multicellular aggregates in normal
morphogenesis and tumor invasion. E-cadherin particularly localizes at cell-cell boundary
regions, and is regulated by integrins 45,46. It increases greatly in concentration during
development from the 2 cell embryo to the blastocyst. In murine E-cadherin-/- embryos,
maternal E-cadherins seem to suffice quantitatively for initial compaction of the morula.
However, when additional, embryo-synthesized cadherin is required for further development,
morphologic polarization of adhesive cells disintegrates 47. Because E-cadherin stabilize cell
adhesions and inhibit cell migration, downregulation and transcriptional loss of this adhesion
molecule is a major mechanism in EMT for both embryonic mesoderm formation and tumor
progression. As an early step in EMT, E-cadherin mediated adhesions of epithelial cells are
loosened to allow for mesenchymal motility. The component of cell-cell junctions, p120
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catenin, may participate in regulation of cell motility by being a link between the formation
and disruption of cadherin-mediated contacts 48. E-cadherin also has growth-suppressive
properties. In tissues lacking E-cadherin, other members of the cadherin family may assume a
similar role. The cell surface receptor tyrosine kinases, ephrins, reversibly signal cell
boundaries in developmental cell recognition, synergize with E-cadherin and, thus, may
likewise be involved in EMT 49. Integrins 50 have an important role in EMT since they
mediate intercellular communication by binding to the ECM and signaling information from
the ECM, through an extensive network of cytoskeletal molecules.
Wnt glycoproteins are a family of secreted signalling molecules that may promote stem cell
renewal, act as hematopoietic growth factors, when abnormally activated they contribute to
carcinogenesis 51, (reviewed in 52) and have a major instructive role in development; Kidney
organogenesis initiates with the condensation of mesenchymal cells and aggregation into a
pretubular clusters that undergo MET to form a simple tubule. This process is impaired in
mice lacking Wnt-4. In such mutant mice, secreted frizzled related protein (sFRP)-2, the Wnt
antagonist, is also absent. Wnt signals are transmitted to the nucleus by β-catenin. Mutations
in β-catenin, lead to defective nephrogenesis and to formation of Wilms’ tumors 53-55. FGF
signalling participates in patterning of mesoderm at gastrulation, through activation of
FGFR1, leading to EMT and morphogenesis of mesoderm at the primitive streak. This is
mediated through β-catenin thus forming a link between FGF and Wnt signalling 56. In
addition, Wnt signalling also occurs independently of β-catenin action 57.
TGFβ1 is a potent inducer of EMT 33,58. Various members of this superfamily are involved in
the regulation of development and in EMT. Additional growth factors are major regulators of
embryonic development (reviewed in 59,60): FGF and hepatocyte growth factor (HGF) are
ligands for tyrosine-kinase receptors. FGFR1 orchestrates EMT and the corresponding
mesodermal morphogenesis by controlling Snail and E-cadherin expression and, probably,
also Brachyury and Tbx6. In FGFR-/- embryos, Wnt3a signalling is attenuated but can be
rescued by lowering E-cadherin levels 56. HGF is a potent modulator of EMT. It activates the
Rho GTPases Cdc42 and Rac, leading to activation of p21-activated kinase (PAK) 61. Cell
motility and branching morphogenesis in EMT are also promoted by members of the EGFCfC gene family, in human and mouse, and are associated with a decrease in β-catenin and an
increased vimentin expression. EGF-CfC activates the ras/raf/mitogen-activated protein
kinase (MAPK) signalling pathways in mammary epithelial cells. Cripto-1, a member of the
EGF-CfC family, is active, e.g., in the embryogenesis of ICM. It also enhances migration and
branching morphogenesis of mouse mammary epithelial cells to undergo EMT 62. Abrogation
of the Cripto gene leads to abnormal mesenchymal development including failure of
gastrulation 63.
An extensively studied example of MET is normal nephron development during conversion of
the metanephric mesenchyme to an epithelial phenotype (reviewed in 64). One of the activator
molecules of MET may be the high mobility group (HMG)-17 protein which is strongly
expressed in the uretric bud where kidney cells start to undergo MET during organogenesis 65.
Cells preparing for MET in the ectoderm of the segmental plate express EphA4, a member of
the ephrin family, which seems to regulate the process of reorganization of the cytoskeleton.
Loss of EphA4 leads to failure of somite formation and irregular kidney morphology 66. In
kidney tubulogenesis, MET is critically dependent on the expression of the developmental
control gene Pax-2 which, in turn, is regulated by TGFβ 67. In metanephric conversion,
alterations of gene transcription have been identified in a multitude of mRNA molecules.
Some examples are cell adhesion molecules syndecan-4 and integrins α6, α3, β1; the
cytokines TGFβ, epidermal growth factor (EGF), fibroblast growth factor (FGF) and bone
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morphogenic protein (BMP) 2, and BMP4 and Neuregulin (Neu-differentiation factor (NDF)
and among transcription factors, β-catenin 68,69.
The various molecules discussed above including ECM, cell surface and soluble cytokines
that mediate EMT and MET, are available both in the embryo and in adult organisms.
Nevertheless, in the adult, the frequency of cellular transitions is very low suggesting that
adult cells either lose their plasticity, or that this property stays intact but is suppressed. We
argue that the experimental data available to date imply this latter possibility. Indeed, adult
tissues contain plastic stem cells, which were identified only following in vitro culture i.e. as a
result of destruction of tissue architecture, or following radiation or chemical induced tissue
damage in vivo. Furthermore, the plastic nature of cells may be unmasked in situation of
tissue damage due to disease 70 or wounding 71.
Expression of E-cadherin is essential for the maintenance of the epithelial phenotype.
Silencing of E-cadherin expression, which is the basis for EMT, involves transcriptional
repression. Several factors have been shown to transcriptionally repress E-cadhrin by binding
to proximal E-boxes in the E-cadherin promoter. These include Snail and Slug, members of a
zinc finger transcription factor family, as well as E47, ZEB-1 and ZEB-2 72-77. Snail and Slug
are highly homologous. However, they differ in the intermediate P-S rich protein region that
contains a 29 amino acid sequence termed the Slug domain. Whereas the role of Snail in EMT
is indisputable, some conflicting information is available, as for the role of Slug. The latter
triggers EMT during chick and Xenopus development whereas in mammalian cells Slug
expression is not always associated with EMT. Nevertheless, recent studies show that Snail
and Slug are functionally equivalent, when examined in the epithelial cell line Madin-Darby
canine Kidney (MDCK), in contributing to EMT and in maintaining the mesenchymal
phenotype 78. Studies in Xenopus further show that Snail lies upstream of Slug in the cascade
leading to neural crest formation and thus these proteins may have distinct functions 79. This
view is supported by the distinct expression patterns of these proteins in development and
during evolution 80. There are also differences in the consequences of knockout of these
genes; Snail-/- embryos form abnormal mesoderm germ layer, are defective in gastrulation
and die early in gestation 81. Mice, mutant for Slug, are viable but show growth retardation 82.
Human breast cancer is associated with elevated expression of the Erb/Neu receptor tyrosine
kinase, which is in fact a prognostic factor indicative of increased progression into invasive
and highly malignant phenotype. Overexpression of constitutively activated Erb/Neu, in
MDCK epithelial cells, causes dissociation of cell-cell contacts and increased motility. In a
tree-dimensional collagen matrix system activated Erb/Neu endowed the cells with a capacity
to form tubules 83.
HGF, mentioned above as a potent inducer of EMT, operates through binding to a tyrosine
kinase receptor, Met. c-Cbl is a downstream target to the Met receptor. Overexpression of cCbl in MDCK cells causes flattening of colonies formed in vitro but no overt EMT occurs. On
the other hand, cells overexpressing a naturally occurring mutant of Cbl, termed 70z-Cbl,
undergo EMT 84. c-Cbl contains multiple protein interaction motifs and serves as a docking
site for many proteins including Crk. Indeed, HGF stimulation causes recruitment of Crk
adaptor proteins into Met dependent signalling complexes. In the absence of HGF stimulation,
overexpression of Crk is sufficient to induce EMT 85.
Members of the Myb family of transcription factors appear to oppose EMT. They promote Ecadherin and integrin mediated cell adhesion to extracellular fiber molecules. Myb proteins
also contribute to maintenance of the state of differentiation of progenitor cells, and are
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involved in apoptosis. Not surprisingly, ICM of B-Myb -/- blastocysts have severely impaired
ES cell proliferation 86.
The traditional concept of cell differentiation would predict that in epithelial cells, genes
encoding for mesenchymal properties are silenced and become reactivated during EMT. At
the same time, genes encoding for epithelial features should be silenced. Although this view is
supported by studies of major epithelial markers, such as E-cadherin, as discussed above, this
may not be case for many cellular proteins, neither in EMT and MET, nor in the case of
plastic behaviour of stem cells, in general. Indeed, mesenchymal cells have been shown to
express, at a low level, T cell receptor (TCR) complex genes 87, and mesenchymal stem cells
express a whole range of markers of lineages into which they potentially may differentiate 88.
Similar promiscuity in gene expression has been demonstrated for HSC 89. Furthermore,
muscle derived stem cells are able to differentiate into hemopoietic progeny while retaining
their myogenic potential 14. It should be considered therefore that a variety of cells, within the
normal cell population, are in a “standby” state, in which they express low levels of
transcripts of different lineages and would readily differentiate upon triggering, without the
requirement of reversal of gene silencing. This promiscuous gene expression pattern may be
the molecular basis for cell plasticity and transdifferentiation and may explain the relative
ease of cell phenotype reprogramming. Indeed, it has been shown that it is sufficient to
overexpress one single transcription factor, in order to shift completely the direction of
differentiation of HSC 90.
Epithelial Mesenchymal Transitions in Tumorigenesis
EMT is believed to be activated during the later stages of tumor progression but does not
seem to play a major role in the initiation of neoplasms. Phenotypic EMT may represent an
early indication that a cell no longer recognizes or respects its neighbours, is no longer subject
to contact inhibition and coordinating synergism, and may serve as a vehicle for tumor
dissemination (Figure 3). Some of the factors promoting EMT are briefly mentioned below.
By contrast to the role of E-cadherin, in maintaining the epithelial phenotype, forced
expression of N-cadherin leads to EMT. The extracellular repeat4 portion of N-cadherin was
shown to be necessary and sufficient to promote EMT and cell motility 91. The transcription
factor HMG-Y, that belongs to the HMGI(Y) family of nonhistone chromatin proteins, seems
to be associated with EMT in metastatic tumor progression of human breast epithelial cells 92.
Figure 3: Tumorigenesis and metastasis entail EMT: A stationary epithelial cell is shown to loose contact with
neighboring cells and to undergo EMT to become a motile, and eventually, metastatic cell.
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Snail triggers EMT associated tumor progression by virtue of its strong suppressor effects on
E-cadherin gene expression 72. Epithelial cells, ectopically expressing snail, adopt a fibroblast
phenotype and acquire tumorigenic and invasive properties. E-cadherin-negative oral
squamous cell carcinomas strongly express Snail. Clinically, Snail is expressed in all ductal
carcinomas with lymph node metastases, including breast tumors. The intensity of Snail
expression correlates inversely with the degree of differentiation 81. Since β-catenin is Ecadherin associated protein and a key component of the Wnt signalling pathway, it is not
surprising that mutated β-catenin is a pathogenetic factors in the transition of pre-malignant to
neoplastic cancer, and in invasion and metastases in several types of cancer 54 (reviewed in
93
). Target genes of β-catenin include matrix metallo-proteinase (MMP)-7, urokinase type
plasminogen activator (uPA), as well as the cell adhesion molecule NrCAM 94. During
promotion of EMT in several pre-malignant and malignant human carcinomas, members of
the EGF/CfC gene family, such as Cripto, suppress β-catenin function. Expression of Cripto
is increased several-fold in EMT, not only in human tumors such as colon, gastric, pancreas,
lung, and breast carcinoma, but is detectable already in pre-malignant lesions of some of these
neoplasms (reviewed in 95). This last observation raises doubts as to whether EMT is indeed
only a late event in the course of carcinogenesis. In fact it has been now shown that cells may
leave the local tumor and disseminate to distant sites were they eventually form methastasis,
early on, before tumor progression in the primary site has been completed 96.
Although TGFβ is a major tumor suppressor cytokine during early stages of tumor
development, blocking cell cycle progression and initial cell growth, many dedifferentiated
late-stage tumors are resistant to this function of TGFβ. They may secrete TGFβ and retain
their susceptibility to TGFβ induced EMT 97,98. TGFβ indeed promotes EMT in late stage
tumors. Of possible clinical significance is a dominant negative type II receptor of TGFβ
(TGFβ-RIIdn) that prevents EMT in EpRas cells. This receptor mutant abolishes completely
the production of metastases in dedifferentiated, highly metastatic mouse colon carcinoma
cells (CT26). In vitro treatment of several human carcinoma cell lines with TGFβ-neutralizing
antibody resulted in loss of invasiveness 99. The capacity of TGFβ to mediate EMT is
dependent on cooperation with Ras signalling; Ha-Ras and TGFβ cause sustained EMT and
metastatic phenotype. This entails activation of Raf/mitogen-activated protein kinase
(MAPK). On the other hand, phosphatidylinositol 3-kinase (PI3K) activation has a
complementary function in promoting e.g. proliferation 100,101. Elevated H-ras expression
induces nuclear accumulation of Smad-2 which promotes EMT and subsequent invasiveness
and metastatic dissemination 102.
EMT may be a reversible phenomenon, even in aggressive tumor cell lines such as small cell
lung cancer, if the cells are allowed to reestablish E-cadherin mediated adhesion contacts.
Transfection with E-cadherin cDNA has a stabilizing effect impairing the capacity of forming
metastases in breast cancer cells 97. Exposure of small cell lung carcinoma to 5-bromodeoxyuridine leads to reversal, back to a normal cell phenotype, with reestablishment of cellcell and cell to matrix adhesions, downregulation of vimentin, upregulation of cytokeratin,
and redistribution of the actin cytoskeleton 34. The elevated degrees of cell plasticity, common
to both embryonal development and tumor progression, suggest, that a principle of tumor
biology could be recapitulation of stages of embryonal development. A comparison between
Snail induced E-cadherin repression in undifferentiated mesoderm and in tissues undergoing
EMT, in early mouse development versus tumor cell lines, lead to the conclusion that “the
same molecules are used to trigger epithelial-mesenchymal transition during embryonic
development and in tumor progression” 72. Wilms tumor is a good example for comparable
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developments in embryonal organogenesis and tumorigenesis. These tumors overexpress
genes corresponding to the earliest stages of metanephric development and underexpress
genes of the later stages, thus mimicking MET 68,69,103. In fact, it has been suggested that
dysregulation of MET could be a major pathogenic factor in the pathogenesis of Wilms’
tumor 53-55.
It is possible that the tumor stroma contains mesenchymally transformed epithelial cancer
cells, generated by EMT, that participate with the physiologic stroma cells in creating a new
tumor’s microenvironment. This issue should be examined along with the possibility that the
tumor mesenchyme might be modified such that it loses some of its “normal” features or,
alternatively, that it acquires new, “transformation” related properties. Although EMT seems
to be closely associated with, and probably is a basic mechanism in cancer progression, more
detailed information is needed on the properties of EMT transformed epithelial cells and on
the sequence of steps that take place during cancer progression.
Summary and Future Perspectives
During development, cells respond to microenvironmental factors by specific phenotypic
alterations that underlay EMT and MET. These dramatic changes, and the subsequent lineage
commitments and formation of the variety of cell types found in methazoa, is the ultimate
form of cell “plasticity”. Recent studies seem to propose that such plastic capabilities may not
be restricted to the embryo but rather, are shared by adult stem cells. Clearly, this issue must
be examined with caution, bearing in mind the limitations of the experimental systems used to
demonstrate stem cell plasticity 104, and the fact that regeneration of the liver, claimed to
result from transdifferentation of HSC, have been now shown to result mainly from cell
fusion 16,17. However, even in this case, the fusion between HSC and hepatocytes results in the
reprogaming of the hemopoietic genome 17, re-emphasizing the plasticity of HSC phenotype.
We reviewed above data that indicate that manifestations of cell plasticity are inheret to
developmental embryonic processes. In the adult organism, plasticity may be less frequent
and, thus, may be subject to tighter restrictions (Figure 4).
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Figure 4: Whereas reversible epithelial mesenchymal transitions (arrows) are the hallmark of embryonic
development, they seem to be markedly restricted, in adult mammalians, by mechanisms which are yet
unresolved.
However, is it biologically concivable that this property is completely disposed off by the
adult organism?. Amphibians have kept remarkable regenerative capacity that entails
plasticity 105. Upon ampuation of a limb or tail in urodete amphibians, a cohort of genes are
co-expressed leading to a process of de-differentation, changes in cell fates and eventually to
the formation of a new organ which is a replica of the lost one 106. We propose that similar
basic rules may apply to any type of regenerative process in the adult organism i.e.,
phenotypic plasticity is an inherent property of cells which they never loose completely.
Indeed, transdifferentiation of pancreas to liver and vice versa has been observed in vivo, in
animal models and in certain human diseases, and was reproduced in vitro (reviewed in 107).
The recent identification of MAPCs point to a possible mechanism for tissue repair through
trnasdifferentation; resident MAPCs may be engaged in tissue maintenance by repairing
micro-defects, due to cell death, that sporadically occur in normal tissues. The discovery of
MAPCs raises further intriguing possibilities: is the capacity of MAPCs to give rise to
epithelial cells mediated through molecular mechanisms of MET? And conversly, do these
cells initially exist in the organism as epithelial cells and convert to MAPCs through EMT?
The gradual reduction in plastic behavior of cells after completion of development suggests,
to our minds, the existence of restraining mechanisms that prevent the expression of this
inherent cellular property, to ensure the stability of already formed tissues and organs. Such
restrictive mode of cell organization has been proposed to account for events within the
hemopoietic microenvironment 20. The blood generating tissue remains in a highly dynamic
form, throughout lifespan, a state that has similarities to embryonic development, and is
accordingly carefully controlled. The “restrictive model of cell organization” suggests that the
microenvironment imposes restrictions of cell growth and differentiation. The exact
12
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localization of cells relative to their environment has critical role in determination of cell fate.
One outstanding example is the importance of orientation in Drosophila male germline stem
cell positioning. These stem cells are arranged oriented to the Hub, somatic cells that form the
“stromal” niche and elaborate renewal signals. Dividing stem cells orient the mitotic spindles
perpendicular to the hub. As a result, one of the daughter cells stays in touch with the hub and
continues to self renew, while the other is pushed away from the niche and subsequently,
differentiates 108. Similarly, the mammalian skin shows a clear organization wherein stem
cells are localized adjacent to mesenchyme, separated by basement membrane. Their
differentiation commences as they depart to distal layers 109. A similar effect of physical
distance of the stem cells from its stromal niche has been suggested for bone marrow stem
cells 110,111.
Apparently, the adult state, in which the restrictions on epithelial mesenchymal transitions are
overtly lifted, is cancer. EMT is a frequent phenomenon in cancer progression and
dissemination. No qualitative differences in signalling from the microenvironment have been
identified with certainty to date, as a cause of cancer progression. However, it has been
suggested that the stroma may transform adjacent epithelial cells, in the absence of preexisting tumor cells (reviewed in 112). Also, some data do suggest, in particular tumors, that
the microenvironment may be altered 113. We suggest that such microenvironmental changes
may exist and should be experimentally tested. Better understanding of restrictive
mechanisms that control tissue organization will provide molecular tools to relieve tissue
restrictions in the adult, to allow for regeneration when needed. Conversely, in tumor states
one would wish to block further cancerous development by re-establishing the lost tissue
restrictions.
Acknowledgments:
D. Zipori is an incumbent of the Joe and Celia Weinstein professorial chair at the Weizmann
Institute of Science. G. Prindull is a Varon Visiting Professor from the University of
Gottingen, Germany. The authors wish to thank Prof. Alexander Bershadsky, from the
Department of Molecular Cell Biology, Weizmann Institute of Science, for critically
reviewing the manuscript.
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Prepublished online January 8, 2004;
doi:10.1182/blood-2003-08-2807
Environmental guidance of normal and tumor cell plasticity: epithelial
mesenchymal transitions as a paradigm
Gregor Prindull and Dov Zipori
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