1 Departamento de Bioquímica, UAM. Instituto de Investigaciones Biomédicas "Alberto Sols" (CSIC-UAM), Arturo Duperier 4, 28029 Madrid, Spain
2 Instituto Cajal CSIC, Doctor Arce, 37, 28002 Madrid, Spain
3 German Cancer Research Center, DKFZ, Im Neuenheimer Feld 280, Heidelberg 69120, Germany
* Author for correspondence (e-mail: acano{at}iib.uam.es)
Accepted 9 February 2004
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Snail, E47, E-cadherin, Invasion, Angiogenesis, Transplantation assays
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Several molecular mechanisms underlying E-cadherin downregulation have been unravelled in the past few years. Among them, hypermethylation of the E-cadherin promoter and transcriptional alterations have emerged as the main mechanisms responsible for E-cadherin downregulation in most carcinomas (Yoshiura et al., 1995; Hennig et al., 1995
; Hajra et al., 1999
; Rodrigo et al., 1999
; Tamura et al., 2000
; Cheng et al., 2001
). Several transcriptional repressors of E-cadherin have been isolated, including the zinc-finger factors Snail (Battle et al., 2000; Cano et al., 2000
) and Slug (Hajra et al., 2002
; Bolós et al., 2003
), the two-handed zinc factors
EF1 (ZEB-1) and SIP-1 (ZEB-2) (Grooteclaes and Frisch, 2000
; Comijn et al., 2001
) and the bHLH factor E12/E47 (Pérez-Moreno et al., 2001
). Snail, Slug and E47 repressors apparently induce a similar phenotype when overexpressed in epithelial MDCK cells, as they elicit a full EMT with all the hallmarks of the process: loss of E-cadherin and other epithelial markers, increased expression and organization of mesenchymal markers, and a motile behaviour (Cano et al., 2000
; Pérez-Moreno et al., 2001
; Bolós et al., 2003
). Furthermore, MDCK cells expressing either Snail or E47 are able to migrate through collagen gels in Boyden-chamber assays and exhibit tumorigenic properties when injected into nude mice (Cano et al., 2000
; Pérez-Moreno et al., 2001
). The involvement of Snail and E47 in tumour progression is also supported by the expression of both genes in carcinoma cell lines with invasive and metastatic properties (Cano et al., 2000
; Pérez-Moreno et al., 2001
; Cheng et al., 2001
; Poser et al., 2001
) and by the expression of Snail in dedifferentiated breast carcinomas and invasive hepatocarcinomas (Blanco et al., 2002
; Sugimachi et al., 2003
). Despite these apparent similarities, the implication of Snail and E47 in specific stages of tumour progression is not yet fully understood. Indeed, several observations suggest that Snail and E47 repressors might play distinct functions. Significant differences in the interaction of both factors with the E-cadherin promoter have been recently detected, with Snail showing a much higher binding affinity (2x1010 M) than E47 (6x109 M) for specific E-boxes (Bolós et al., 2003
). In addition, Snail and E2A (encoding E12/E47) exhibit distinct expression patterns in early embryonic development of mammals; Snail is expressed at EMT areas (Cano et al., 2000
; Carver et al., 2001
; Locascio et al., 2002
) whereas E2A is expressed in the already migratory mesodermal cells (Pérez-Moreno et al., 2001
).
To gain further insight into the role of Snail and E47 in tumour progression, we used previously characterized MDCK-Snail and MDCK-E47 cell lines (Cano et al., 2000; Pérez-Moreno et al., 2001
; Bolós et al., 2003
) and performed an in-depth analysis of their properties when grown in vitro in organotypic cultures, as well as in vivo in transplantation assays and xenografted tumours.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transplantation assays
MDCK cells were precultured on collagen I gels in DMEM. They were covered with a silicon transplantation chamber (Renner, Gmbh) and transplanted onto the dorsal muscle fascia of 6-week-old male BALB/c nude mice, following the published protocol (Boukamp et al., 1990; Skobe et al., 1997
). Eight mice were used for each MDCK cell line. Animals were maintained under sterile conditions and observed every 2 days. Four animals from each group were sacrificed after 10 and 20 days of transplantation. Transplants were resected and half were embedded in OCT and frozen in liquid nitrogen for cryostat sectioning and immunofluorescence with the remainder fixed in formaldehyde and embedded in paraffin as described above.
Induction of xenografted tumours
Cells grown in two-dimensional cultures were trypsinized, washed and resuspended at a density of 1x107 cells/ml in PBS. 1x106 cells of the indicated cell lines were injected into the flanks of 8-week old male BALB/c nude mice (Charles River). Mice were maintained under sterile conditions and observed every two days. The size and growth of the tumours were estimated from the external diameter measured with a calibre, and the mice sacrificed when the tumours reached a size of 1.5 cm of larger external diameter. Control mice (injected with MDCK-CMV cells) were observed for up two months. Tumours were excised and immediately frozen in isopentane-cooled liquid nitrogen embedded in OCT. All animal experiments were performed according to institutional guidelines for animal care. A total of ten tumours from MDCK-E47 transfectants were generated from a single clone and 12 tumours were generated from two independent clones of MDCK-Snail transfectants (six tumours each). At least four different tumours derived from each cell line were analysed by histology, immunostaining and in situ hybridization.
Immunofluorescence analysis
Sections (5-10 µm) of the OCT-embedded samples from either the organotypic cultures, in vivo transplants or xenografted tumours were fixed in methanol (20°C) and acetone (20°C) and then incubated with the primary antibodies (Navarro et al., 1993; Cano et al., 2000
). The primary antibodies included: rat monoclonal anti-mouse E-cadherin (1:100) (ECCD2) (a gift of M. Takeichi, Kyoto University, Japan), mouse monoclonal anti-vimentin (1:100) (Dako), anti-cytokeratin 8 (1:20) (Progen, Heidelberg, Germany), anti-endoglin (1:100) (South. Biotech. Associates) and rat monoclonal anti-mouse CD31 (1:100) (BD Pharmingen). Secondary antibodies included goat anti-rat and goat anti-mouse Ig coupled to Alexa 594 and Alexa 488 respectively (1:1000) (Molecular Probes). Samples from the organotypic cultures and transplants were also stained with DAPI to detect nuclei.
In situ hybridization analysis
Vibratome sections at 40-100 µm were obtained from the OCT specimens. After elimination of the OCT compound by washing in 4% paraformaldehyde, sections were subjected to in situ hybridization as previously described (Blanco et al., 2002). The riboprobes labelled with digoxygenine nucleotides used were: full length mouse E-cadherin, Snail and E47 cDNA, as previously described (Cano et al., 2000
; Pérez-Moreno et al., 2001
); and full length mouse VEGF-A and TGFß-1 cDNA, kindly provided by F. Larcher (CIEMAT, Madrid, Spain) and M. Quintanilla (IIB. Madrid, Spain), respectively. Some sections were processed for immunohistochemistry with a monoclonal antibody for
-smooth muscle actin (Sigma), following treatment with peroxidase-coupled secondary antibody and developing with DAB-H2O2.
Proliferation assays
To monitor proliferation, cells were seeded in triplicate onto 96-well plates at a density of 5000 cells/well. After 24 hours of growth in normal medium, radiolabelled [3H]thymidine (0.5 µCi) was added and the cells were grown for an additional 24 hours. Cells were harvested and [3H] radioactivity was measured in a solid scintillation counter.
Quantification of migrated cells in collagen gels and markers in xenografted tumours
Sections prepared for immunofluorescence analysis obtained from 10-day organotypic cultures and transplantation assays were analysed to score the number of cells invading the collagen gel both from the upper part (MDCK infiltrated cells) and lower part of the cultures (emigrated host stromal cells). Quantification was performed by calculating the percentage of the distance migrated into the collagen type I gel by either cell type in relation to the total gel thickness. At least four sections of each culture and cell type were analysed and the data presented as the average. Internal variation among the different samples of each cell type was less than 10%. Quantification of E-cadherin/Snail/E47 expression in xenografted tumours was estimated on in situ hybridization sections by calculating the number of positive cells for each of the marker on the overall surface section. Quantification of CD31 positive cells in MDCK-Snail and MDCK-E47 induced tumours was also performed on immunofluorescence sections.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
These results indicate that MDCK-Snail cells have a 2- to 3-fold higher infiltrative potential in organotypic cultures than MDCK-E47 cells, and this behaviour seems to be an intrinsic property of the cells. They also suggest that sustained expression of Snail is required for induction of the invasive behaviour of mesenchymal cells under these experimental conditions, since its loss in the uppermost layer is associated with an epithelial non-invading phenotype.
MDCK-Snail and MDCK-E47 cells have distinct invasive behaviour in transplantation assays
The in vivo invasive potential of Snail- and E47-expressing cells was analysed in surface transplantation assays. Cells were seeded on the top of collagen type I matrices that were then transplanted onto the back muscle fascia of nude mice, as previously described for keratinocyte cultures (Boukamp et al., 1990; Skobe et al., 1997
). The transplants were allowed to grow for up to 10 and 20 days when mice were sacrificed and analysis performed. After 10 days of culture, transplants from control MDCK-CMV cells grew as an organized epithelial multilayer (Fig. 2Aa,Ba) with apparently differentiated cyst-like structures (Fig. 2Aa, black arrowheads). MDCK-CMV cells were unable to infiltrate the adjacent collagen type I gel (Fig. 2Aa,Ba). In agreement with this phenotype, E-cadherin expression was detected at cell-cell contacts in most cells of the 10-day control MDCK transplants (Fig. 2Ad,Bd). Co-expression of vimentin was detected in some isolated cells of the deepest layer of 10-day control transplants (Fig. 2Bd). Transplants of MDCK-Snail cells grown for 10 days form an undifferentiated multilayer with infiltration of the deeper cells up to around 45% of the total collagen type I gel thickness (Fig. 2Ab,Bb, black arrowheads). No expression of E-cadherin was noted but homogeneous expression of vimentin was detected in all cells of the 10-day MDCK-Snail transplants (Fig. 2Ae,Be). The 10-day transplants of MDCK-E47 cells grew as undifferentiated multilayers (Fig. 2Ac,Bc) which maintain the repression of E-cadherin and homogenous expression of vimentin (Fig. 2Af,Bf). However, only 15% of the collagen type I gel was infiltrated in the superficial part by MDCK-E47 cells (Fig. 2Ac,Bc, black arrowheads). In contrast, the lower part of the collagen type I gel (in direct contact with the host) of 10-day MDCK-E47 transplants shows clear accumulation of vimentin-positive cells (Fig. 2Ac,f, red arrows). These cells have apparently migrated from the host stromal tissue and infiltrated around 55% of the gel's thickness. A similar situation was detected in the lower part of the collagen gel in the 10-day MDCK-Snail transplants, although the gel was only invaded from the bottom to about 10-15% of its thickness (Fig. 2Ab,e, red arrows). By contrast, no migrated cells were observed in that region of the 10-day MDCK control transplants (Fig. 2Aa,d, red arrows).
|
Analysis after 20 days of in vivo culture showed that control MDCK transplants were overgrown as a highly organized multilayer of differentiated epithelial cells with an abundance of cyst-like structures that were unable to infiltrate the remaining collagen type I gel (Fig. 3Aa,d). Indeed MDCK-CMV cells clearly display a delimitation of the seeded cells from the collagen gel, suggesting that they have organized a basal membrane (Fig. 3Ad and data not shown; yellow arrow in panel d indicates basal membrane delimitation). Immunofluorescence analysis of parallel transplants with a less hyperplastic epithelium showed E-cadherin expression at cell-cell contacts of all cell layers of 20-day control MDCK transplants (Fig. 3Ba), which also showed complete absence of vimentin expression (Fig. 3Bd). This indicates that MDCK-CMV cells fully differentiate under these experimental conditions, losing the residual expression of mesenchymal markers observed in the 10-day transplants. The 20-day transplants of MDCK-Snail and MDCK-E47 cells showed an overgrowth of highly undifferentiated cells that completely infiltrated the whole collagen type I gel and the stromal host tissues up to the muscle and adipose tissues (Fig. 3Ab,c,e,f and data not shown). As previously observed in the organotypic cultures, the 20-day cultures of MDCK-Snail transplants showed a transdifferentiation to an epithelial-like phenotype in the uppermost layers, characterized by expression of CK-8 (data not shown) and E-cadherin at cell-cell contacts and the absence of vimentin (Fig. 3Bb,e, white arrows). Co-expression of E-cadherin and vimentin was observed in some cells at the interface between the epithelial and mesenchymal layers (Fig. 3Be, white arrowheads), indicative of an intermediate phenotype. As observed in the organotypic cultures, 20-day transplants of MDCK-E47 cells maintained the undifferentiated phenotype in all cell layers (Fig. 3Ac,f), as confirmed by the complete absence of E-cadherin and homogenous expression of vimentin even in the uppermost cell layers (Fig. 3Bc,f).
|
MDCK-Snail and MDCK-E47 cells exhibit a distinct in vivo angiogenic potential
The detection of vimentin-positive cells in the lower part of the collagen gel in 10-day transplants of MDCK-E47 and to a lesser extent in those of MDCK-Snail cells (see Fig. 2A), together with the absence of a clear boundary between the collagen gel and the host tissue (Fig. 2A, dotted lines), suggest that these vimentin-expressing cells may represent mesenchymal cells that have migrated from the host stroma. However, it is also possible that other cell types from the host tissue, such as endothelial cells, are also migrating into the collagen gel. To examine this aspect further, double immunofluorescence staining for the CD31 endothelial marker and vimentin were performed on the 10-day transplants. As can be observed in Fig. 4, adjacent sections of the MDCK-Snail and MDCK-E47 transplants shown in Fig. 2 exhibited CD31 staining in all the host stroma in close contact with the collagen gel edge (Fig. 4e,f, yellow arrows). In contrast, only a few CD31-positive cells (around 10-20% of the total host volume tissue) were detected in the stroma of control MDCK transplants, and these cells were localized far away from the lower collagen gel edge. Furthermore, hardly any vimentin or CD31-positive cells were observed inside the upper zone of the collagen gel in control transplants (Fig. 4a,d). Interestingly, although few CD31-positive cells appeared to be migrating into the collagen gel of the MDCK-Snail transplants (Fig. 4e) (colonizing 5-10% of the collagen gel in its lower portion), a large number of CD31-positive cells were detected infiltrating (from the bottom up) about 50% of the collagen gel in MDCK-E47 transplants (Fig. 4f, top level of CD31 positive cells indicated by red arrows). In addition, some blood vessel-like structures were detected in the host stromal compartment of MDCK-Snail and MDCK-E47 transplants even expanding into the colonized region of the collagen gel in MDCK-E47 transplants (Fig. 4h,i, white arrows). The blood vessel-like structures detected in much deeper regions of the stromal compartment of control MDCK transplants (Fig. 4d,g) are likely to be the normal blood vessels of the host tissue. Similar results were obtained in other samples from independent transplants of each cell type. The above results strongly suggest that MDCK-Snail and particularly MDCK-E47 cells are inducing an angiogenic response of the host stromal tissue under these experimental conditions.
|
The angiogenic response was more obvious after 20 days of culture when clear infiltration of blood vessels into the implanted MDCK-Snail and MDCK-E47 cells was observed (Fig. 5b,c, blue arrows). In marked contrast were MDCK control transplants in which the vessels are restricted to the host stroma (Fig. 5a,d, blue arrows). A detailed observation of the histological sections revealed that the angiogenic response of MDCK-Snail and MDCK-E47 cells extends all over the transplants, since sprouting blood vessels reached the uppermost external regions of the implants (Fig. 5e,f, blue arrows). However, the angiogenic response of the control MDCK cells was restricted to the stromal tissue (Fig. 5d, blue arrow). The in vivo malignant behaviour of MDCK-Snail and MDCK-E47 cells was also correlated in the 20-day transplants with a total invasion of the host stroma and reabsorption of the implanted collagen gel detected (Fig. 5b,c, yellow arrows indicate the hypothetical edge of collagen gel). In contrast, in control MDCK transplants, the collagen gel largely persists and the implanted cells maintain the epithelial phenotype and a clear basal membrane-like delimitation without invading the gel (Fig. 5a, yellow arrow indicates the edge of collagen gel).
|
The results presented so far indicate that MDCK-Snail and MDCK-E47 cells exhibit distinct in vitro and in vivo behaviour when grown as cultures. Snail-expressing cells are more likely to invade the collagen gel at short incubation periods than E47 expressing cells, exhibiting a 2- to 3-fold increase in their invasion capacity. Although both cell types induce an angiogenic response of the host tissue, this property is approximately 3- to 4-fold higher in MDCK-E47 cells.
Tumorigenic properties of MDCK-Snail and MDCK-E47 cells.
MDCK-Snail and MDCK-E47 cells are tumorigenic when injected into nude mice (Cano et al., 2000; Pérez-Moreno et al., 2001
). The different behaviour exhibited by each cell type in organotypic cultures and transplantation assays, led us to carry out a closer examination of their in vivo properties when growing in nude mice. Additional injection studies into nude mice confirmed previous results, with both cell types inducing tumours at all injection sites. However, the tumours induced by MDCK-E47 cells grew much faster than those induced by MDCK-Snail cells (Fig. 6A). Analysis of the tumour growth dynamics demonstrated that MDCK-E47 transfectants show a 2-fold higher proliferation potential than MDCK-Snail transfectants (Fig. 6A). Proliferation assays performed in two-dimensional cultures also showed that MDCK-E47 cells grow between 1.6- and 2-fold faster than MDCK-Snail cells in in vitro culture conditions (Fig. 6B).
|
Xenografted tumours induced by both MDCK-Snail and MDCK-E47 cells were subjected to histological, immunofluorescence and ISH analysis. At least four different tumours generated by each cell line were studied, and representative examples are presented in Fig. 6C. Tumours induced by MDCK-Snail and MDCK-E47 cells are highly undifferentiated spindle cell tumours (Fig. 6Ca,f), although one of the MDCK-Snail-induced tumours showed a 20-30% of the total area with apparent differentiation (data not shown). The degree of differentiation of tumours was confirmed by immunostaining for E-cadherin. No E-cadherin expression was observed in the undifferentiated tumours induced by either cell type (Fig. 6Cb,g), in contrast to the normal expression of E-cadherin at cell-cell contacts of the epidermis adjacent to the tumours (Fig. 6Cb, inset). ISH analysis showed complete absence of E-cadherin mRNA in the undifferentiated tumours induced by MDCK-Snail cells (Fig. 6Cc) which showed strong expression of Snail transcripts in 80% of all tumour cells (see detail of an inner area of the tumour in Fig. 6Cd). Surprisingly, a diffuse but consistent expression of E-cadherin mRNA was observed in undifferentiated tumours induced by MDCK-E47 cells (Fig. 6Ch); these showed a `salt and pepper' pattern with expression of E47 mRNA in around 30-40% of tumour cells (see detail of an inner area of the tumour in Fig. 6Cj). As expected, the areas of MDCK-E47 tumours with high levels of E47 transcripts seem to correspond to those with complete absence of E-cadherin mRNA, indicating an effective repression of E-cadherin transcription in the cells with active expression of the E47 repressor. Interestingly, ISH of MDCK-Snail tumours also showed expression of E47 mRNA in some scattered cells (Fig. 6Ce), which might coincide with those showing stronger Snail expression (Fig. 6C, compare panels d and e). No expression of Snail mRNA was detected in the MDCK-E47 induced tumours (Fig. 6Ci). These results suggest that Snail can induce E47 in restricted areas of tumours, whereas the reverse situation does not occur.
The angiogenic capacity of the tumours induced by MDCK-Snail and MDCK-E47 cells was also analysed by staining with the endothelial markers CD31 and endoglin. As shown in Fig. 7A, both kinds of tumour contained abundant endothelial cells co-expressing both markers that organized into blood vessel-like structures. However, tumours induced by MDCK-E47 cells contain more blood vessels (Fig. 7Ae,f) of smaller size compared to those induced by MDCK-Snail cells (Fig. 7Aa,b). The differences in blood vessel organization could also be detected by immunohistochemical staining of -smooth muscle actin (
SMA) in both kinds of tumour (Fig. 7Ac,d,g,h). Quantitative estimation of the vessel density of the different tumours was performed by confocal analysis of serial sections stained with the CD31 marker. This study showed that tumours induced by MCDK-E47 cells have a higher CD31 staining density (3500 CD31 units/mm2 surface) than those induced by MDCK-Snail cells (1400 CD31 units/mm2 surface). Two-dimensional reconstitution of the images obtained from the confocal serial analyses showed that MDCK-E47-induced tumours did contain a very high density of small CD31-positive vessels covering most of the tumour surface. Tumours induced by MCDK-Snail cells have a lower density of larger CD31 vessels that are restricted to the inner area of the tumours (data not shown). These results indicate that tumours induced by both MDCK-Snail and MDCK-E47 cells have a high angiogenic potential, although they have different organization and/or size of blood vessels. Analysis of pro-angiogenic factors VEGF-A and TGFß1 was performed on both types of tumours by ISH. MCDK-Snail and MDCK-E47 induced tumours showed expression of TGFß1 transcripts in a rather scattered pattern across the extent of the tumour (Fig. 7Ba,c). MDCK-Snail-induced tumours also showed expression of VEGF-A transcripts in isolated patches that were also positive for
SMA, although this ectopic expression did not correspond to morphologically visible blood vessels (Fig. 7Bb). These results indicate that proangiogenic factors are produced by tumours induced by MDCK-Snail and MCDK-E47 cells.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In vitro organotypic cultures and in vivo transplantation assays of MDCK cells stably expressing Snail or E47 repressors clearly indicate that MCDK-Snail cells have stronger infiltration capacity than MDCK-E47 cells over short time periods. Organotypic cultures showed 20% and transplantation assays 45% invasion of the gel layer by MDCK-Snail cells. In contrast, only 5-10% of the gel surface was invaded by MDCK-E47 cells in either experimental setting after 1 week or 10 days of culture. After a longer period in culture, MDCK-Snail cells continued to infiltrate into the collagen gel, but MCDK-E47 cells seem to be more restrained from invasion, particularly in the organotypic cultures. MCDK-Snail cells that infiltrate the collagen gel maintain the mesenchymal-like phenotype, as characterized by the loss of epithelial markers E-cadherin and CK-8 and high expression of vimentin. However, MDCK-Snail cells in the uppermost non-invading layers showed a clear transdifferentiation to an epithelial-like phenotype with re-expression of E-cadherin organized in cell-cell contacts, in both organotypic cultures and transplantation assays (Fig. 1 and Fig. 3B). The transdifferentiation behaviour of MDCK-Snail cells has never been observed in two-dimensional culture conditions, where the cells remain as a homogenous population of mesenchymal-like cells after prolonged passage. This suggests that either the host environment or the tri-dimensional organization of the cells is responsible for the transdifferentiation observed.
Interestingly, the mesenchymal-like cells which maintain Snail expression are those able to invade the collagen gel in both organotypic cultures and transplantation assays, whereas the epithelial-like cells that have lost Snail expression are non-invasive. These results strongly suggest that sustained expression of Snail is required for induction of invasiveness, and are in full agreement with the phenotype of Snail null mice, which are unable to undergo EMT (Carver et al., 2001). This situation is also reminiscent of the transient expression of Snail observed in specific developmental contexts in mammals. Expression is associated with the regions undergoing EMT but silenced in some developmental stages once cells are already migrating or when they suffer the reverse mesenchymal to epithelial transition (Sefton et al., 1998
; Nieto, 2002
). In the context of development, Snail expression seems to require both inductive and maintenance signals provided by different growth factors, such as TGFß and FGFs, respectively (Thiery, 2002
; Nieto, 2002
; Ciruna and Rossant, 2001
). The loss of Snail expression observed in the uppermost layer of the organotypic cultures and transplants of MDCK-Snail cells after longer times of culture suggest the absence or weakness of maintenance signals in those regions. Additional mechanisms affecting to the stability of Snail mRNA in those experimental conditions could also explain the observed expression pattern.
Since Snail expression is driven by the CMV promoter, mechanisms such as those affecting the stability of Snail mRNA may account for the loss of Snail expression observed in the uppermost layer of the organotypic cultures and transplants of MDCK-Snail cells after longer times of culture. This hypothesis is supported by the fact that the Snail cDNA expressed in stable transfections carries the original 3'UTR region of Snail mRNA (Cano et al., 2000). Therefore, Snail mRNA degradation can be promoted by an as yet unidentified mechanism in the upper levels of organotypic culture under a cell compaction environment. Intra-clonal variations of Snail expression may also explain the observed Snail downregulation in the upper layers of the cultures. However, the fact that MDCK-E-47 cells have more intra-clonal E-47 expression variability with a clear `salt and pepper' pattern, whereas transdifferentiation does not occur in MDCK-E47 cells at any time of culture under either experimental situation, makes this possibility highly unlikely.
Although further studies are needed to clarify this specific aspect, the present results suggest that EMT induced by Snail might be reversible, at least in some specific cell or tissue contexts. Alteration of mRNA stability could also explain the heterogeneous expression of E47 transcripts observed in both kinds of culture of MCDK-E47 cells. Nevertheless, and in contrast to MDCK-Snail cells, MDCK-E47 cells maintain the mesenchymal phenotype and the absence of E-cadherin protein in all culture conditions either in in vitro three-dimensional organotypic cultures, in in vivo transplant assays and in the xenografted tumours. In spite of this, MDCK-E47 cells have a lower invasive capacity than MDCK-Snail cells at short incubation periods, suggesting that the EMT mediated by E47 is not sufficient to induce a highly invasive potential and may require a proper environment to develop the invasion process. Indeed, after long culture periods, sustained expression of E47 is able to confer invasiveness, since the 3-week transplants of MDCK-E47 cells exhibit an in vivo infiltrating potential similar to that induced by MDCK-Snail transplants (see Fig. 3A and Fig. 5). These observations support the idea that besides E-cadherin repression, Snail and E47 must regulate additional genes (as primary or secondary targets) whose expression or repression might be required to provide cells with a high invasion potential. In this context, it is worthy of note that local or transient expression of Snail in a few cells might be sufficient to produce local invasion, an event that will initiate the escape of tumour cells and the metastatic cascade. The local and restricted expression of Snail at EMT regions in the mouse embryo (Sefton et al., 1998; Cano et al., 2000
; Carver et al., 2001
) and, more importantly in tumours induced in the mouse skin, as well as in human breast carcinomas and hepatocarcinomas (Cano et al., 2000
; Blanco et al., 2002
; Sugimachi et al., 2003
) fully supports this idea. Interestingly, the MDCK-Snail-induced tumours showed a focal expression of E47 apparently coincident with regions of stronger Snail expression, indicating a potential cooperation of both factors in specific in vivo contexts. On the other hand, focal E-cadherin mRNA expression could be detected in MDCK-E47 induced tumours that, nevertheless exhibited a complete absence of E-cadherin protein, indicating additional levels of regulation of E-cadherin in this specific tumoral context. Post-transcriptional regulation of E-cadherin affecting protein stability might be one of the mechanisms involved. Indeed, our previous studies on ectopic expression of E-cadherin in dedifferentiated spindle carcinoma cells showed that E-cadherin turnover was highly increased, rendering the cells unable to express high levels of the ectopic protein or to organize it in defined cell-cell contacts, despite the high levels of exogenous E-cadherin mRNA expression (Navarro et al., 1993
; Lozano and Cano, 1998
). Additional studies are required to clarify this specific aspect.
An interesting and unexpected observation was made in the transplantation assays: the induction of a migratory response of host cells into the collagen gel. At short time periods (1 week of culture) this response was much stronger in the transplants of MDCK-E47 than in those of MDCK-Snail cells. Interestingly, a significant proportion of the migrated cells from the host stroma are endothelial cells, determined by staining with the CD31 marker, and have the ability to organize themselves into blood vessel-like structures in the host tissue in proximity to the transplant and within the collagen gel. Angiogenesis is defined as the formation of new capillaries from existing vessels. Although this process occurs in physiological conditions during embryonic development and wound healing, it is crucial in tumour progression. Indeed, tumour cells release angiogenic factors or repress the synthesis of inhibitors. Blood vessels were particularly evident in the MDCK-E47 transplants (Fig. 4). However, after prolonged culture (3 weeks) a strong and widespread angiogenic response is observed in the transplants of both cell types. These observations indicate that a strong angiogenic response of the host tissue is induced by MDCK-E47 cells and to a lesser extent by MDCK-Snail cells under the transplant assay conditions. Indeed, analysis of xenografted tumours induced by both kinds of cell indicates that they are highly angiogenic, with a higher density of endothelial vessels being detected in the MDCK-E47 than in MDCK-Snail-induced tumours. These differential angiogenic properties might be, at least in part, responsible for the distinct proliferation capacity of the tumours. Thus, the higher in vivo angiogenic capacity of MDCK-E47 cells compared to MDCK-Snail cells might contribute to the higher proliferation potential of the tumours induced by the first cell type. However, other effects exerted by both factors on cell proliferation and/or cell survival, can not be discounted at present.
In this context, elucidation of the angiogenic and migratory factors induced by either cell type or the host stroma will be required to fully understand the role of Snail and E47 in tumour progression. Candidates for the induction of angiogenesis and cell migration during development and tumour progression include members of the TGFß, FGF and VEGF families. Indeed, TGFß-1 and FGFs are involved in the regulation of Snail expression (Spagnolli et al., 2000; Ciruna and Rossant, 2001
; Valdés et al., 2002
; Yañez-Mo et al., 2003
; Peinado et al., 2003
) and are strong candidates for roles in tumour progression and migration. In agreement with this, TGFß-1 and VEGF-A expression has been detected in the MDCK-Snail tumours, while MDCK-E47 tumours seem to preferentially express TGFß-1 transcripts at the analysed time points (Fig. 7B). These observations do not discount expression of other members of the VEGF family and/or additional angiogenic factors by MDCK-E47 cells at different time periods of in vivo growth. In fact, our ongoing studies on expression profiling in MDCK-Snail and MDCK-E47 cells indicate differential induction of several pro-angiogenic factors in both cell types such as VEGF, jagged1 and nocth2 (H.P., G. Moreno-Bueno, E.C., D. Sarrio, S. Villa, V. Bolós, J. Palacios and A.C., unpublished). Interestingly, jagged1 has been implicated in angiogenesis during development through activation of Notch signalling (Shimizu et al., 1999
; Shimizu et al., 2000
). In this context, a recent study (Morel et. al., 2003
) proposes that Snail can downregulate negative regulators of Notch signalling during development, providing a possible link between those molecules in the control of gene expression. These observations suggest that Snail and E47 are directly or indirectly involved in the regulation of several genes implicated in tumour progression and angiogenesis.
The present results suggest differential and distinct roles of Snail and E47 in in vivo invasiveness and tumour progression. Snail has a prominent role in the promotion of local invasion and E47 acts to maintain a dedifferentiated and migratory phenotype contributing to a strong angiogenic response from the host stromal tissue. These results, therefore, provide new information of interest to the design of therapeutic strategies for blocking specific stages of tumour progression, such as local invasion and angiogenesis.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Batlle, E., Sancho, E., Franci, C., Dominguez, D., Monfar, M., Baulida, J. and Garcia de Herreros, A. (2000). The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat. Cell Biol. 2, 84-89.[CrossRef][Medline]
Behrens, J., Mareel, M. M., van Roy, F. M. and Birchmeier, W. (1989). Dissecting tumor cell invasion: epithelial cells acquire invasive properties after loss of uvomorulin-mediated cell-cell adhesion. J. Cell Biol. 108, 2435-2447.[Abstract]
Bergers, G. and Benjamin, L. E. (2003). Tumorigenesis and the angiogenic switch. Nat. Rev. Cancer 3, 401-410.[CrossRef][Medline]
Berx, G., Cleton-Jansen, A. M., Nollet, F., de Leeuw, W. J., van de Vijver, M., Cornelisse, C. and van Roy, F. (1995). E-cadherin is a tumour/invasion suppressor gene mutated in human lobular breast cancers. EMBO J. 14, 6107-6115.[Abstract]
Birchmeier, W. and Behrens, J. (1994). Cadherin expression in carcinomas: role in the formation of cell junctions and the prevention of invasiveness. Biochim. Biophys. Acta Mol. Cell Res. 1198, 11-26.[CrossRef][Medline]
Blanco, M. J., Moreno-Bueno, G., Sarrio, D., Locascio, A., Cano, A., Palacios, J. and Nieto, M. A. (2002). Correlation of Snail expression with histological grade and lymph node status in breast carcinomas. Oncogene 21, 3241-3246.[CrossRef][Medline]
Bolós, V., Peinado, H., Pérez-Moreno, M. A., Fraga, M. A., Esteller, M. and Cano, A. (2003). The transcription factor Slug represses E-cadherin expression and induces epithelial to mesenchymal transitions: a comparison with Snail and E47 repressors. J. Cell. Sci. 116, 499-511.
Boukamp, P., Stanbridge, E. J., Foo, D. Y., Cerutti, P. A. and Fusenig, N. E. (1990). c-Ha-ras oncogene expression in immortalized human keratinocytes (HaCaT) alters growth potential in vivo but lacks correlation with malignancy. Cancer Res. 50, 2840-2847.[Abstract]
Boyer, B., Valles, A. M. and Edme, N. (2000). Induction and regulation of epithelial to mesenchymal transitions. Biochem. Pharmacol. 60, 1091-1099.[CrossRef][Medline]
Burdsal, C. A., Damsky, C. H. and Pedersen, R. A. (1993). The role of E-cadherin and integrins in mesoderm differentiation and migration at the mammalian primitive streak. Development 118, 829-844.
Cano, A., Pérez-Moreno, M. A., Rodrigo, I., Locascio, A., Blanco, M. J., del Barrio, M. G., Portillo, F. and Nieto, M. A. (2000). The transcription factor Snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nat. Cell Biol. 2, 76-83.[CrossRef][Medline]
Carver, E. A., Jiang, R., Lan, Y., Oram, K. F. and Gridley, T. (2001). The mouse snail gene encodes a key regulator of the epithelial-mesenchymal transition. Mol. Cell. Biol. 21, 8184-8188.
Cheng, C. W., Wu, P. E., Yu, J. C., Huang, C. S., Yue, C. T., Wu, C. W. and Shen, C. Y. (2001). Mechanisms of inactivation of E-cadherin in breast carcinoma: modification of the two-hit hypothesis of tumor suppressor gene. Oncogene 20, 3814-3823.[CrossRef][Medline]
Christofori, G. and Semb, H. (1999). The role of the cell-adhesion molecule E-cadherin as a tumour-suppressor gene. Trends Biochem. Sci. 24, 73-76.[CrossRef][Medline]
Ciruna, B. and Rossant, J. (2001). FGF signalling regulates mesoderm cell fate specification and morphogenetic movement at the primitive streak. Dev. Cell 1, 37-49.[Medline]
Comijn, J., Berx, G., Vermassen, P., Verschueren, K., van Grunsven, L., Bruyneel, E., Mareel, M., Huylebroeck, D. and van Roy, F. (2001). The two-handed E box binding zinc finger protein SIP1 downregulates E-cadherin and induces invasion. Mol. Cell 7, 1267-1278.[CrossRef][Medline]
Grooteclaes, M. L. and Frisch, S. M. (2000). Evidence for a function of CtBP in epithelial gene regulation and anoikis. Oncogene 19, 3823-3828.[CrossRef][Medline]
Hajra, K. M., Ji, X. and Fearon, E. R. (1999). Extinction of E-cadherin expression in breast cancer via a dominant repression pathway acting on proximal promoter elements. Oncogene 18, 7274-7279.[CrossRef][Medline]
Hajra, K. M., Chen, D. Y. and Fearon, E. R. (2002). The SLUG zinc-finger protein represses E-cadherin in breast cancer. Cancer Res. 62, 1613-1618.
Hanahan, D. and Weinberg, R. A. (2000). The hallmarks of cancer. Cell 100, 57-70.[Medline]
Hay, E. D. (1995). An overview of epithelio-mesenchymal transformation. Acta Anat. 154, 8-20.[Medline]
Hennig, G., Behrens, J., Truss, M., Frisch, S., Reichmann, E. and Birchmeier, W. (1995). Progression of carcinoma cells is associated with alterations in chromatin structure and factor binding at the E-cadherin promoter in vivo. Oncogene 11, 475-484.[Medline]
Locascio, A., Manzanares, M., Blanco, M. J. and Nieto, M. A. (2002). Modularity and reshuffling of Snail and Slug expression during vertebrate evolution. Proc. Natl. Acad. Sci. USA 99, 16841-16846.
Lozano, E. and Cano, A. (1998). Induction of mutual stabilization and retardation of tumor growth by coexpression of plakoglobin and E-cadherin in mouse skin spindle carcinoma cells. Mol. Carcinog. 21, 273-287.[CrossRef][Medline]
Maas-Szabowski, N., Stark, H. J. and Fusenig, N. E. (2000). Keratinocyte growth regulation in defined organotypic cultures through IL-1-induced keratinocyte growth factor expression in resting fibroblasts. J. Invest. Dermatol. 114, 1075-1084.
Morel, V., le Borgne, R. and Schweisguth, F. (2003). Snail is required for Delta endocytosis and Notch-dependent activation of single-minded expression. Dev. Genes. Evol. 213, 65-72.[Medline]
Navarro, P., Lozano, E. and Cano, A. (1993). Expression of E- or P-cadherin is not sufficient to modify the morphology and the tumorigenic behaviour of murine spindle carcinoma cells. Possible involvement of plakoglobin. J. Cell Sci. 105, 923-934.
Nieto, M. A. (2002). The snail superfamily of zinc-finger transcription factors. Nat. Rev. Mol. Cell. Biol. 3, 155-166.[CrossRef][Medline]
Peinado, H., Quintanilla, M. and Cano, A. (2003). Transforming growth factor ß-1 induces Snail transcription factor in epithelial cell lines. Mechanisms for epithelial mesenchymal transitions. J. Biol. Chem. 278, 21113-21123.
Pérez-Moreno, M. A., Locascio, A., Rodrigo, I., Dhondt, G., Portillo, F., Nieto, M. A. and Cano, A. (2001). A new role for E12/E47 in the repression of E-cadherin expression and epithelial-mesenchymal transitions. J. Biol. Chem. 276, 27424-27431.
Perl, A. K., Wilgenbus, P., Dahl, U., Semb, H. and Christofori, G. (1998). A causal role for E-cadherin in the transition from adenoma to carcinoma. Nature 392, 190-193.[CrossRef][Medline]
Poser, I., Dominguez, D., García de Herreros, A., Varnai, A., Buettner, R. and Bosserhoff, A. K. (2001). Loss of E-cadherin expression in melanoma cells involves up-regulation of the transcriptional repressor Snail. J. Biol. Chem. 276, 24661-24666.
Rodrigo, I., Cato, A. C. and Cano, A. (1999). Regulation of E-cadherin gene expression during tumor progression: the role of a new Ets-binding site and the E-pal element. Exp. Cell. Res. 248, 358-371.[CrossRef][Medline]
Sefton, M., Sanchez, S. and Nieto, M. A. (1998). Conserved and divergent roles for members of the Snail family of transcription factors in the chick and mouse embryo. Development 125, 3111-3121.
Shimizu, K., Chiba, S., Kumano, K., Hosoya, N., Takahashi, T., Kanda, Y., Hamada, Y., Yazaki, Y. and Hirai, H. (1999). Mouse jagged1 physically interacts with notch2 and other notch receptors. Assessment by quantitative methods. J. Biol. Chem. 274, 32961-32969.
Shimizu, K., Chiba, S., Hosoya, N., Kumano, K., Saito, T., Kurokawa, M., Kanda, Y., Hamada, Y. and Hirai, H. (2000). Binding of Delta1, Jagged1, and Jagged2 to Notch2 rapidly induces cleavage, nuclear translocation, and hyperphosphorylation of Notch2. Mol. Cell. Biol. 20, 6913-6922.
Skobe, M., Rockwell, P., Goldstein, N., Vosseler, S. and Fusenig, N. E. (1997). Halting angiogenesis suppresses carcinoma cell invasion. Nat. Medicine 3, 1222-1227.[Medline]
Spagnolli, F. M., Cichini, C., Tripodi, M. and Weiss, M, C. (2000). Inhibition of MMH (Met murine hepatocyte) cell differentiation by TGF-ß is abrogated by pre-treatment with heritable differentiation effector FGF1. J. Cell. Sci. 113, 3639-3647.
Stark, H. J., Baur, M., Breitkreutz, D., Mirancea, N. and Fusenig, N. E. (1999). Organotypic keratinocyte cocultures in defined medium with regular epidermal morphogenesis and differentiation. J. Invest. Dermatol. 112, 681-691.
Sugimachi, K., Tanaka, S., Kameyama, T., Taguchi, K., Aishima, S., Shimada, M., Sugimachi, K. and Tsuneyoshi, M. (2003). Transcriptional repressor Snail and progression of human hepatocellular carcinoma. Clin. Cancer Res. 9, 2657-2664.
Takeichi, M. (1993). Cadherins in cancer: implications for invasion and metastasis. Curr. Opin. Cell. Biol. 5, 806-811.[Medline]
Tamura, G., Yin, J., Wang, S., Fleisher, A. S., Zou, T., Abraham, J. M., Kong, D., Smolinski, K. N., Wilson, K. T., James, S. P. et al. (2000). E-Cadherin gene promoter hypermethylation in primary human gastric carcinomas. J. Natl. Cancer Inst. 92, 569-573.
Thiery, J. P. (2002). Epithelial-mesenchymal transitions in tumor progresión. Nat. Rev. Cancer 2, 442-454.[CrossRef][Medline]
Valdes, F., Alvarez, A. M., Locascio, A., Vega, S., Herrera, B., Fernandez, M., Benito, M., Nieto, M. A. and Fabregat, I. (2002). The epithelial mesenchymal transition confers resistance to the apoptotic effects of transforming growth factor beta in fetal rat hepatocyres. Mol. Cancer Res. 1, 68-78.
Yañez-Mo, M., Lara-Pezzi, E., Selgas, R., Ramirez-Huesca, M., Dominguez-Jimenez, C., Jimenez-Heffernan, J. A., Aguilera, A., Sanchez-Tomero, J. A., Bajo, M. A., Alvarez, V. et al. (2003). Peritoneal dialysis and epithelial-to-mesenchymal transtition of mesothelial cells. N. Engl. J. Med. 348, 403-413.
Yokoyama, K., Kamata, N., Hayashi, E., Hoteiya, T., Ueda, N., Fujimoto, R. and Nagayama, M. (2001). Reverse correlation of E-cadherin and snail expression in oral squamous cell carcinoma cells in vitro. Oral Oncol. 37, 65-71.[CrossRef][Medline]
Yoshiura, K., Kanai, Y., Ochiai, A., Shimoyama, Y., Sugimura, T. and Hirohashi, S. (1995). Silencing of the E-cadherin invasion-suppressor gene by CpG methylation in human carcinomas. Proc. Natl. Acad. Sci. USA 92, 7416-7419.[Abstract]