Instituto de Neurociencias de Alicante CSIC-UMH, Apartado 18, Sant Joan d'Alacant, 03550, Spain
* Author for correspondence (e-mail: anieto{at}umh.es)
SUMMARY
The functions of the Snail family of zinc-finger transcription factors are essential during embryonic development. One of their best-known functions is to induce epithelial to mesenchymal transitions (EMTs), which convert epithelial cells into migratory mesenchymal cells. In recent years, many orthologues of the Snail family have been identified throughout the animal kingdom, and their study is providing new clues about the EMT-dependent and -independent functions of Snail proteins. Here, we discuss these functions and how they influence cell behaviour during development and during diseases such as metastatic cancer. From these findings, we propose that Snail genes act primarily as survival factors and inducers of cell movement, rather than as inducers of EMT or cell fate.
Introduction
The function that Snail genes are best known for is the induction of a
phenotypic change called epithelial to mesenchymal transition (EMT).
Snail-induced EMT converts epithelial cells into mesenchymal cells with
migratory properties that contribute to the formation of many tissues during
embryonic development and to the acquisition of invasive properties in
epithelial tumours. Snail-induced EMT is partly due to the direct repression
of E-cadherin transcription both during development and tumour
progression. As the loss of E-Cadherin expression in tumours is considered to
be a marker of a poor clinical outcome, E-Cadherin repressors are regarded as
markers of malignancy, and as targets for anti-invasive drugs (for reviews,
see Nieto, 2002;
Thiery, 2002
).
Snail genes also have additional cellular functions that sometimes occur independently of the induction of EMT. They protect cells from the death induced either by the loss of survival factors or by direct apoptotic stimuli. In some instances, survival properties emerge concomitant with the induction of EMT, whereas, in others, both the resistance to cell death and the persistence of the epithelial phenotype are required. In addition, there are certain cell movement processes that do not require a full EMT, such as mesoderm formation in Drosophila and in anamniotic vertebrate embryos (vertebrate embryos that lack an amniotic membrane, such as amphibian and fish embryos). Interestingly, recent evidence shows that Snail genes also participate in these processes by regulating cell adhesion and migration. Thus, it seems that the prevalent function of Snail might be to regulate cell adhesion rather than to induce EMT. In this context, the triggering of the EMT would be just one of the mechanisms used by these transcription factors to allow cell movement.
Another commonly discussed function of Snail genes is their role as mesodermal determinants. Again, recent data from both invertebrates and vertebrates indicate that their participation in mesoderm formation is more related to their role in regulating cell movement than to their putative function as inducers of cell fate.
Here, we review recent insights into the evolution of the Snail superfamily, and discuss a new unified nomenclature for this family of transcription factors that will facilitate the comparison of family members among distant species. We also discuss recent data on how Snail family members are post-transcriptionally regulated. Finally, on the basis of recent findings, we propose a new model of Snail function, in which Snail genes act as survival factors and as inducers of cell movement, rather than as inducers of mesodermal fate or EMT. This new theory will be discussed within the context of the properties that both embryonic and tumour cells share when they become migratory and invasive.
The Snail superfamily: new members and new nomenclature
snail was first described in Drosophila melanogaster
(Boulay et al., 1987), where it
was shown to be essential for the formation of the mesoderm
(Alberga et al., 1991
). In the
20 or so years since its isolation, more than 50 family members have been
described in metazoans (reviewed by Nieto,
2002
) (see below also), with several family members found in
different groups.
During metazoan evolution, the generation of gene families is believed to
have occurred by gene duplication and by the divergence of an ancestral gene
(Ohno, 1970;
Ohno, 1999
;
Furlong and Holland, 2002
).
Until recently, it was assumed that it was independent gene duplications that
gave rise to the four snail genes in insects (snail, escargot, worniu
and scratch), and to the two in vertebrates (Snail and
Slug). However, from the results of database searching and
phylogenetic analyses, it was proposed a few years ago that the Snail
superfamily consists of two related, but independent, families: Snail
and Scratch (Manzanares et al.,
2001
; Nieto,
2002
). (See Fig. 1
for an updated version of the Snail family phylogenetic tree, including all
the members isolated in the last four years.) The phylogeny shown in
Fig. 1 is compatible with a
revised evolutionary history that has been previously proposed
(Nieto, 2002
) and which is
updated in Fig. 2. According to
this phylogenetic history, the duplication of a single snail gene in
the metazoan ancestor gave rise to two genes, snail and
scratch, which, after undergoing independent duplications in the
different Cnidaria and Bilateralia, themselves gave rise to a number of genes
in each group (Fig. 2).
|
Snail genes: mesodermal determinants or inducers of cell movement?
The evolutionary origin of the mesoderm is still a matter of debate
(reviewed by Technau and Scholz,
2003). In order to resolve this question, the homologues of
so-called `mesodermal determinant' genes have been isolated in diploblastic
animals (animals with only two germ layers ectoderm and endoderm),
such as in the Cnidarians. Snail is regarded as one of the
interesting genes to study due to its classification as a mesodermal
determinant gene. This classification explains why, in the last few years,
Snail homologues have been identified in Cnidarians, such as the anemone
Nemastostella vectensis
(Fritzenwanker et al., 2004
;
Martindale et al., 2004
), the
coral Acropora millepora (Hayward
et al., 2004
) and the jellyfish Podocoryne carnea
(Spring et al., 2002
).
|
An analysis of Snail expression and function in triploblasts (animals with
three germ layers), among representatives of the Lophotrochozoans, Ecdysozoans
and Deuterostomes, supports a role for Snail in regulating cell movement. For
instance, both in the mollusc Patella vulgata
(Lespinet et al., 2002) and in
the spider Achaearanea tepidariorum
(Yamazaki et al., 2005
),
snail homologues are not expressed in the mesoderm, but rather in ectodermal
tissues that undergo changes in cell shape or morphogenetic movements.
Similarly, Snail is expressed in the developing skin of the mouse when skin
cells lose E-cadherin expression and invaginate to form the hair follicule
buds (Jamora et al., 2005
).
Furthermore, it is worth noting that Snail-mutant mice die at gastrulation
because of defects in the EMT, which is needed in amniotes for mesoderm
development (Carver et al.,
2001
). Interestingly, mesoderm forms in these mutants and
expresses well-known mesodermal markers, such as brachyury, despite being
unable to downregulate E-cadherin and migrate. To conclude, these data
together therefore suggest that Snail regulates cell movement and adhesion
rather than cell fate.
Snail genes at the crossroads of the EMT
The first indication that the Snail gene family had a role in triggering
EMT came from Snail2 loss-of-function experiments carried out in
chick embryos (Nieto et al.,
1994); a role that was later confirmed in cell lines and in other
vertebrate embryos (reviewed by Nieto,
2002
; De Craene et al.,
2005
). EMT is crucial for the formation of many different tissues
and organs during embryogenesis, such as for the development of the mesoderm
in amniotes, the neural crest in all vertebrates, as well as the heart
cushions and the palate, among others. Interestingly, Snail genes are
expressed in all EMT processes where they have been studied (reviewed by
Nieto, 2002
). EMT can be
triggered by different signalling molecules, such as by epidermal growth
factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF),
transforming growth factor ß (TGFß), bone morphogenetic proteins
(BMPs), WNTs and Notch. In agreement with the involvement of Snail in all
studied processes of EMT, these signalling molecules have been shown to induce
Snail genes in different cellular contexts (see
Fig. 3 for a summary of the
signalling pathways that can activate Snail genes) (for a review, see
De Craene et al., 2005
).
With respect to the TGFß superfamily, TGFß1 induces
Snail1 in hepatocytes (Spagnoli
et al., 2000; Valdés et
al., 2002
), in the palate
(Martinez-Alvarez et al.,
2004
) and in epithelial
(Peinado et al., 2003
) and
mesothelial cells (Yáñez-Mo
et al., 2003
; Margetts et al.,
2005
). TGFß2 induces Snail1 in the developing mouse
skin (Jamora et al., 2005
) and
Snail2 during heart development (Romano and Ruyan, 2000); and BMP4
induces Snail2 during neural crest development
(Dickinson et al., 1995
;
Liem et al., 1995
).
EGF is also linked to the induction of Snail and EMT in several ways. It
reduces E-Cadherin function by promoting its caveolin-dependent endocytosis,
which leads to subsequent Snail induction and to the triggering of EMT
(Lu et al., 2003). Another
recent connection between EGF, Snail and EMT has been described in mice that
express the EGF family member Crypto in the mammary gland. A full EMT process
accompanied by the induction of Snail expression is observed both in the
hyperplasias and tumours derived from these mice
(Strizzi et al., 2004
).
Furthermore, the EGFR pathway can activate STAT3, which can upregulate Snail
function via activation of the zinc-finger transporter LIV1
(Yamashita et al., 2004
),
which has also been associated with the progression of breast tumours
(Manning et al., 1994
). In
this work, LIV1 was described as being a target of the estrogen receptor (ER)
pathway. Interestingly, recent data have also related this signalling pathway
with Snail expression (Fujita et al.,
2003
). The metastasis-associated protein 3 (MTA3) is an ER target
that has been identified as being a component of the repressor complex that
directly inhibits Snail transcription in breast epithelial cells.
Indeed, the absence of ER signalling or MTA3 leads to aberrant Snail
expression and EMT. This could, at least in part, explain why a poor prognosis
is associated with ER-negative breast tumours
(Lapidus et al., 1998
).
|
In this section, we have only highlighted some recent data regarding the numerous extracellular factors that induce the expression of Snail family members in the context of EMT. The conclusion that can be drawn is that Snail gene induction is a central convergence point for the factors that induce this cellular event.
EMT-related and EMT-independent Snail functions
Although Snail seems to be required for all processes of EMT that have been studied, this does not necessarily mean that the induction of EMT is the prevalent role of Snail genes. As such, they have additional roles that sometimes operate independently of the induction of EMT, which we discuss below.
One EMT-independent role that is fulfilled by all Snail superfamily members
is the protection of cells from cell death. In C. elegans, the
repression of the Scratch homologue, CES-1
(Metzstein and Horvitz, 1999),
promotes the physiological death of a particular class of neurons. In humans,
a translocation converts the repressor hepatic leukemic factor (HLF; a
putative CES-2 homologue) into an activator that, in turn, induces Snail2 and
leads to aberrant cell survival and to the development of leukaemia
(Inukai et al., 1999
).
Furthermore, haematopoietic progenitors in Snail2 null-mutant mice
show an increased sensitivity to death induced by gamma-irradiation
(Inoue et al., 2002
;
Pérez-Losada et al.,
2003
). Snail1 is also a potent survival factor:
Snail1-expressing cells survive being deprived of survival factors;
are resistant to the action of direct apoptotic stimuli that signal through
the death receptor; and are resistant to DNA damage
(Kajita et al., 2004
;
Martinez-Alvarez et al., 2004
;
Vega et al., 2004
).
In some circumstances, the survival properties that are conferred to cells
by the expression of the Snail genes are acquired concomitantly with the
induction of EMT, as in fetal hepatocytes
(Spagnoli et al., 2000;
Gotzmann et al., 2002
;
Valdés at al., 2002
)
and the neural crest (Vega et al.,
2004
; Tribulo et al.,
2004
). However, in the palate, Snail-induced resistance to cell
death occurs despite the epithelial architecture of the tissue remaining
intact. The Snail-mediated survival of the epithelial cells that lie at the
medial edge of the developing palate takes place when the two palatal shelves
do not fuse. This occurs in some pathological situations, for example in cleft
palate defects in mammals, and where cleft palates have evolved as the normal
condition, such as in avians
(Martinez-Alvarez et al.,
2004
). Interestingly, Snail1 is the family member involved in this
process in the mouse and Snail2 plays this role in the chick. This is another
example of the functional interchange that has been described for the family
members during evolution in other processes, such as mesoderm and neural crest
development (Locascio et al.,
2002
).
The cleft palate condition reveals a situation in which Snail genes are
expressed and are active in epithelial cells and still do not induce EMT or
changes in cell adhesion. The mechanism by which EMT induction is prevented in
this case is not known, and as one can always evoke differences in cellular
context, it will be extremely interesting to decipher these differences. They
could be related to the absence of Snail co-factors or of particular Snail
downstream targets. This point takes us back to the origin of the neural
crest. This is because Snail is expressed in the dorsal region of the
neural tube in non-vertebrate chordates (ascidians and amphioxus)
(Langeland et al., 1998;
Wada and Saiga, 1999
), where
neural crest cells form in vertebrates. However, ascidian and amphioxus do not
have a proper neural crest. Interestingly enough, it has been recently
reported that in the ascidian embryo, some cells from the anterior part of the
neural tube do have migratory abilities characteristic of neural crest cells
(Jeffery et al., 2004
). These
cells might thus represent the link between neural crest specification and the
acquisition of migratory behaviour in vertebrates, providing us with clues
about the evolutionary origin of the neural crest (see
Manzanares and Nieto, 2003
).
Again, as in the case of cleft palate formation, it would be interesting to
know why EMT does not occur in these cells. It could be that certain Snail
downstream targets have been recruited only in the vertebrate lineage.
Alternatively, if amphioxus snail-expressing cells could migrate, it
is possible that the environment does not provide the necessary clues to
trigger this process. Nevertheless, there is no evidence that snail is fully
active in the neural tube of pre-vertebrates. In the absence of good
anti-snail antibodies, the possibility that the snail protein is not
translated or is maintained in an inactive state by post-translational
mechanisms (see below) in these organisms cannot be excluded. However, we know
that Snail genes are active in the cleft palate situation at least to induce
cell survival, making it an excellent model in which to study
Snail-dependent/EMT-independent processes
Snail genes as cell-adhesion regulators
In addition to finding processes that are governed by Snail independently
of the induction of EMT, different experimental models have revealed that
Snail functions in some cell movements that do not require a full EMT.
Interestingly, recent evidence shows that Snail genes also participate in
these processes by regulating cell adhesion and migration
(Yamashita et al., 2004;
Savagner et al., 2005
). One
example of this is mesoderm formation in anamniote vertebrate embryos (such as
amphibia and fish). During mesoderm formation in amniotes, cells delaminate
and migrate individually. However, in anamniotes, a complex interplay of
different morphogenetic movements causes a mass, sheet-like migration of
cells, in which cells maintain contact with each other while moving
(Keller et al., 2000
).
Ironically, it is the lack of a bona fide EMT during mesoderm formation in
Xenopus and zebrafish that might have made some investigators
reluctant to consider the Snail genes as being important players in
convergence and extension movements (reviewed by
Locascio and Nieto, 2001
).
However, very recently, snail1a has been implicated in the anterior
movement of the axial mesendoderm in the zebrafish embryo
(Yamashita et al., 2004
).
Although the mechanism underlying its function in this process is not yet
understood, it seems clear that LIV1 is necessary for the nuclear localisation
of Snail1a and that its downregulation induces defects in anterior mesoderm
migration in zebrafish (Yamashita et al.,
2004
). Curiously enough, and possibly due to the implication of
snail, the authors mention that EMT is impaired by both liv1 and
snail1a misexpression. As already discussed, mesendoderm migration
does not occur through a bona fide EMT, but still, Snail1a is important for
the extension movement. Our own data on the other snail1 gene in
zebrafish (snail1b) extend and reinforce this idea (M. J. Blanco, A.
B.-G., A. E. Reyes, M. Tada, M. Allende, S. W. Wilson, R. Mayor and M. A. N.,
unpublished).
The idea that Snail is involved in the movement of cells that maintain
contact with each other as they move is consistent with its role during
gastrulation in Drosophila. In the fly, snail is expressed
in the cells of the ventral furrow that invaginate and give rise to the
mesoderm (Leptin, 1991). In
this context, again, the changes in cell shape that accompany the
morphogenetic movements do not require a full EMT. Cells move together even
though Snail still functions as an E-Cadherin repressor. Cell-cell adhesion is
reduced but maintained due to a switch in expression from E- to N-Cadherin
(Oda et al., 1998
). At the
cellular level, a similar process occurs during mesoderm formation in
ascidians (Wada and Saiga,
1999
) and hair bud formation in mice
(Jamora et al., 2005
). As
already mentioned, Snail1 is expressed during the invagination of the
hair bud precursor cells, which occurs concomitantly with E-Cadherin
downregulation.
Another example in which Snail genes participate in cell movements that do
not require a full EMT has been recently published
(Savagner et al., 2005). This
study found that Snail2 is involved in the re-epithelialisation of cutaneous
wounds in mice, a process that requires migration and reduced cell-cell
adhesion, but in which the cells involved retain intercellular junctions and
remain associated with each other in a cohesive sheet
(Savagner et al., 2005
).
During this process, there is no upregulation of Snail1 expression,
which is reflected in the maintained expression of E-cadherin in the margins
of the wound and which supports the idea that Snail2 is a much weaker
repressor of E-cadherin than Snail1
(Bolós et al., 2003
). It
will be interesting to identify the adhesion molecules that Snail2 regulates
in this process. Future work should also aim to identify all the targets that
are directly or indirectly regulated by Snail genes and which are in common or
specific to each family member. Taking into account that similar roles are
carried out by Snail1 and Snail2 in different species
(Nieto, 2002
), the search for
these targets and their specificity is going to be a difficult task, although
undoubtedly extremely interesting. With respect to targets,
Fig. 4 summarises those that
are involved in processes in which Snail genes: repress epithelial markers
(Cano et al., 2000
;
Batlle et al., 2000
;
Guaita et al., 2002
;
Ikenouchi et al., 2003
;
Tripathi et al., 2005a
);
upregulate mesenchymal markers (Cano et
al., 2000
; Guaita et al.,
2002
); or participate in the change of cell shape and invasive
properties (del Barrio and Nieto,
2002
; Yokoyama et al.,
2003
), cell proliferation
(Vega et al., 2004
) or cell
survival (Kajita et al., 2004
;
Tribulo et al., 2004
;
Vega et al., 2004
).
|
Controlling Snail activity by subcellular localisation
It has been recently shown in vitro that the activity of the Snail1 protein
is regulated by phosphorylation, which, in turn, regulates its subcellular
localisation (Domínguez et al.,
2003; Zhou et al.,
2004
; Yook et al.,
2005
). Current data suggest that exportins (such as CRM1), which
control the translocation of proteins from the nucleus to the cytoplasm, are
involved in exporting phosphorylated Snail1 and, thus, in its inactivation as
a transcription factor (Domínguez
et al., 2003
). Interestingly, one of the kinases that
phosphorylates Snail1 is GSK3, which not only promotes the nuclear export of
Snail1 but also its rapid degradation via the proteasome
(Zhou et al., 2004
;
Yook et al., 2005
)
(Fig. 3). Thus, the
phosphorylation of Snail1 exquisitely controls its activity. These data are
also compatible with the finding that active GSK3, which maintains Snail1 in
an inactive state, is required to prevent an EMT from occurring in breast and
skin epithelial cell lines. In these cells, inhibition of GSK3 activity also
induces Snail transcription, adding a new mechanism by which GSK3
regulates Snail1 (Bachelder et al.,
2005
). In addition to GSK3, the p21-activated kinase (PAK1) is
also able to phosphorylate Snail at a different residue and to control its
subcellular localisation. Interestingly, PAK1-induced phosphorylation favours
the nuclear localisation of Snail and, thus, its activity as a transcription
factor (Yang et al.,
2005
).
The relationship of Snail1 with GSK3 connects Snail to the WNT signalling
pathway. Indeed, in the presence of WNT signalling, GSK3 is unable to
phosphorylate its targets and thus, both ß-catenin and Snail1 are
stabilised and ready to act as transcription factors. ß-catenin itself,
acting as a transcription factor through its interaction with TCF/LEF, is
required for EMT both in epithelial cells
(Kim et al., 2002) and during
heart cushion development (Liebner et al.,
2004
). These data suggest that cooperation occurs between WNT
signalling and other Snail-induced signalling pathways, such as FGF, in the
triggering of the EMT. This cooperation has been already highlighted in
several developmental systems, in particular, in the mesoderm and the neural
crest (Ciruna et al., 2001; Bastidas et
al., 2004
; Meulemans and
Bronner-Fraser, 2004
). For example, when Snail1 activity is
maintained, E-cadherin is repressed and is therefore not available to bind
ß-catenin and form adherens junctions. As a result, ß-catenin is
available to bind to TCF/LEF and to act as a transcription factor, promoting
WNT signalling. Although this situation will only occur concomitantly with an
inactive ß-catenin degradation system, WNT signalling can increase Snail1
function by preventing its nuclear export and degradation, and Snail1 can
promote WNT signalling by keeping E-cadherin downregulated.
LIV1 also appears to regulate Snail function by controlling it by an as-yet
unknown mechanism (Yamashita et al.,
2004). Thus, GSK3 and LIV1 might play opposite roles,
downregulating and activating the function of Snail, respectively
(Fig. 3).
In addition to being regulated at the transcriptional level, the data discussed above indicate that Snail function is also regulated by its subcellular localisation. Indeed, Snail is a pleiotropic protein that needs to be tightly regulated, as its misexpression is detrimental in many ways, as discussed below.
Snail functions: development versus pathology
One conclusion that can be drawn from the numerous recent studies of the Snail proteins is that they mainly function to regulate cell movement and to provide cells with survival properties. While these functions are crucial for embryonic development, they become fatal in pathological situations in the adult. The Snail-mediated induction of cell movements is translated in the embryo into the ability to generate different tissues and organs that are located far from where their precursors originate. In cancer, however, they facilitate the delamination of cells from the primary tumour and their metastasis to other parts of the body (Fig. 5).
|
Snail2 has also been recently added to the list of Snail family members
that are involved in tumour progression
(Uchikado et al., 2005). This
study's finding that SNAIL2 is downregulated in human esophageal squamous
carcinomas suggests that Snail2, in addition to Snail1, can also induce
pathological EMT to occur in particular cell types or to cooperate with Snail1
in this process. An example of this co-operation might occur in human breast
tumour cells, where SNAIL1 expression has been correlated with
dedifferentiation and metastasis (Cheng et
al., 2001
; Blanco et al.,
2002
; Elloul et al.,
2005
), and SNAIL2 expression with the repression of the tumour
suppressor gene BRCA2 (Tripathi
et al., 2005b
). Snail2 is also activated in malignant
mesotheliomas, where it is induced by stem cell factor (SCF)
(Catalano et al., 2004
), which
is in agreement with its previous description as a target of the SCF/c-Kit
pathway (Pérez-Losada et al.,
2002
). In these tumours, SNAIL2 expression seems to be
associated with a patient's resistance to chemotherapeutic agents, in keeping
with the described role of Snail2 in protecting cells from cell death
(Inoue et al., 2002
;
Pérez-Losada et al.,
2003
; Kajita et al.,
2004
).
There are circumstances in which the dissemination of a primary tumour
involves its exposure to low levels of oxygen, which helps it to acquire
malignant properties. A recent study has shown that hypoxia indeed induces
SNAIL1 expression and invasiveness in ovarian carcinoma cells
(Imai et al., 2003). Although
the mechanism for Snail induction is not yet understood, a hint may come from
pancreatic cancer cells, in which hypoxia induces the transcription of the
autocrine motility factor (AMF; Fig.
3), a protein that can act both as a cytoplasmic enzyme and as an
extracellular cytokine. AMF, in turn, induces Snail and can generate liver
metastasis when overexpressed in cells orthotopically administered to nude
mice (Tsutsumi et al.,
2004
).
In summary, advances in the last few years have lead to our understanding that Snail1 in particular, and the Snail genes in general, are new potential targets of anti-invasive drugs, owing to their association with dedifferentiated metastatic tumours of different origins.
Fibrosis and wound healing
Pathological EMT is not only observed during tumour progression, but also
in other circumstances, such as fibrosis. Thus, it not surprising to find that
SNAIL1 is activated in the mesothelial cells of patients during the secondary
fibrosis that is associated with prolonged peritoneal dialysis
(Yáñez-Mo et al.,
2003). TGFß1 induces the same effects both in cultured human
mesothelial cells (Yáñez-Mo
et al., 2003
) and in rats that receive an intraperitoneal
injection of an adenovirus vector that transfers active TGFß1
(Margetts et al., 2005
). EMT
and Snail1 induction is also observed after the incubation of mesothelial
cells with menstrual effluent (Demir et
al., 2004
).
Fibrosis also appears in the kidney concomitant with Snail1 expression
after injury, and TGFß can also mimic this effect both in vitro and in
vivo (Sato et al., 2003).
Snail expression may be a general response to injury in epithelial cells, as
Snail2 is activated during skin wound healing, as already mentioned
(Savagner et al., 2005
), and
Snail1 appears during cataract extraction in lens epithelial cells that
concomitantly undergo EMT (Saika et al.,
2004
).
Snail effects on cell proliferation and survival in development and disease
There is one more theme to consider regarding the conversion of cells from
an epithelial to a mesenchymal phenotype. This conversion gives rise to cells
that resemble fibroblasts, which, intuitively, one would expect to have an
increased proliferation rate. However, cell division is impaired in
Snail-expressing epithelial cells that have undergone EMT
(Valdés et al., 2002;
Peinado et al., 2003
;
Vega et al., 2004
). For
example, changes in cell shape concur with low proliferation and the
expression of Snail in several systems, such as in the premigratory neural
crest in chick and mouse (Burstyn-Cohen and
Kalcheim, 2002
; Vega et al.,
2004
), in the ventral furrow during Drosophila
gastrulation (Foe, 1989
) and
in the invasive front of carcinomas (Jung
et al., 2001
). This observation is striking because tumour
development is usually associated with increased cell proliferation. However,
it is worth considering that the change to an invasive phenotype is related to
a tumour's acquisition of malignant properties, not with its formation or
growth. Thus, Snail would favour invasion versus tumour growth
(Fig. 5). Invasion is also
favoured by the angiogenic properties of Snail
(Peinado et al., 2004
).
During hair bud formation, Snail-expression correlates with E-cadherin
downregulation and increased proliferation
(Jamora et al., 2005).
Interestingly, in this system, cells maintain the epithelial phenotype. Thus,
decreased cell proliferation could be linked to the profound reorganization of
the cytoskeleton that occurs concomitantly with the EMT, and which may be
incompatible with a highly proliferative state. Alternatively, it could depend
on cellular context and might be related to the absence of some Snail targets
or co-factors. The latter is consistent with the finding that particular
Drosophila snail mutant alleles show an intermediate phenotype in the
cells that normally express the gene. These cells express both mesodermal and
ectodermal markers, suggesting that the regulation of different targets is
independently affected in these mutants
(Hemavathy et al., 1997
).
As discussed above, during embryonic development, Snail gene expression
protects certain cell populations from cell death, such as the neural crest
(Vega et al., 2004;
Tribulo et al., 2004
), and the
epithelial cells at the medial edge of the palate when the palate fails to
fuse (Martinez-Alvarez, 2004). In a mouse model of colonic neoplasia, the
min mouse, Snail downregulation by antisense oligonucleotides has
been shown to increase cell death in colon tumours
(Roy et al., 2004
), confirming
its role in cell survival in cancer. In this model, tumour incidence also
decreased with Snail downregulation and the tumours showed decreased
proliferation rates.
These findings thus show that the acquisition of movement, survival and invasive properties by Snail-expressing cells that delaminate from the mesoderm or the neural tube in developing embryos, or from a primary tumour in an adult, gives these cells a selective advantage and ability to travel considerable distances. Such cells might stop being exposed to survival factors that are present in their tissue of origin as they move away from this tissue, and might also encounter apoptotic factors during their migration through hostile territories. If Snail proteins indeed protect migrating cells from death, they would thus allow embryonic migratory cells to reach their final destinations, while unfortunately also disseminating malignant cells through an adult to give rise to metastasis (Fig. 5).
Conclusion
It is clear that future research will provide much more information on the functions of this gene family both in development and in disease. It will be important to identify endogenous repressors to understand how embryonic cells stop migrating when they reach their target tissues and also synthetic inhibitors to specifically interfere with the delamination of cells from primary tumours. It is possible that Snail inactivation could help to prevent invasiveness and help in making invasive cells more susceptible to destruction. Thus, hopefully, future advances in our understanding of the Snail gene family's mechanisms of action and regulation will help us to gain insights into essential developmental pathways and into one of the worst sides of cancer, the onset of the metastatic process.
ACKNOWLEDGMENTS
We are grateful to all members of M. A. Nieto's laboratory for encouraging discussions. Work in the laboratory is mainly supported by grants from the Spanish Ministry of Education and Science. A.B.-G. is a researcher of the Ramon y Cajal Programme (MEC).
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