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Human Molecular Genetics Advance Access originally published online on October 5, 2005
Human Molecular Genetics 2005 14(22):3449-3461; doi:10.1093/hmg/ddi373
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Cancer development induced by graded expression of Snail in mice

Pedro Antonio Pérez-Mancera1,{dagger}, María Pérez-Caro1,{dagger}, Inés González-Herrero1, Teresa Flores2, Alberto Orfao3, A. Garcia de Herreros4, Alfonso Gutiérrez-Adán5, Belén Pintado5, Ana Sagrera1, Manuel Sánchez-Martín1,6 and Isidro Sánchez-García1,*

1Laboratorio 13, Instituto de Biología Molecular y Celular del Cáncer (IBMCC), CSIC/Universidad de Salamanca, Campus Unamuno, 37007 Salamanca, Spain, 2Servicio de Anatomía Patológica and 3Servicio de Citometría, University of Salamanca, Salamanca, Spain, 4Unitat de Biologia Cellular i Molecular, Institut Municipal d'Investigacio Medica, Universitat Pompeu Fabra, Barcelona, Spain, 5Area de Reproducción Animal, Centro de Investigación y Tecnología, Ctra de la Coruña km 5.9, 28040 Madrid, Spain and 6Departamento de Medicina, Universidad de Salamanca, Salamanca, Spain

* To whom correspondence should be addressed. Tel: +34 923238403; Fax: +34 923294813; Email: isg{at}usal.es

Received August 18, 2005; Accepted September 29, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The zinc-finger transcription factor Snail is believed to trigger epithelial–mesenchymal transitions (EMTs) during cancer progression. This idea is supported by analysis of Snail knockout mice, which uncovered crucial role of Snail in gastrulation, and of individuals with cancer, in whom Snail expression is frequently upregulated. However, these results have not shown a direct link between Snail and the pathogenesis of cancer. Here we show that mice carrying hypomorphic tetracycline-repressible Snail transgenes, that increase Snail expression to 20% above normal levels, exhibit no morphological alterations and develop both epithelial and mesenchymal tumours (leukaemias). Suppression of the Snail transgene did not rescue the malignant phenotype, indicating that alterations induced by Snail are irreversible. CombitTA-Snail murine embryonic fibroblasts show similar migratory ability to that of control mouse embryonic fibroblasts (MEFs). However, CombitTA-Snail-MEFs induce tumour formation in nude mice. CombitTA-Snail expression results in increased radioprotection in vivo, although it does not affect p53 regulation in response to DNA damage. In concert with these results, Snail expression is repressed following DNA damage. This regulation of Snail by DNA damage is p53-independent. Our results connect DNA damage with the requirement of a critical level of an EMT regulator and provide genetic evidence that Snail plays essential roles in cancer development in mammals and thereby influences cell fate in the genotoxic stress response.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Snail family of zinc-finger transcription factors occupies a central role for mesoderm formation in several organisms from flies to mammals (1Go). The first member of the Snail family, snail, was described in Drosophila melanogaster (2Go,3Go), where it was shown to be essential for the formation of mesoderm (4Go). The transfection of Snail in mammalian epithelial cells (5Go,6Go) and the phenotype of the Snail mutant mice, where it is essential for gastrulation (7Go), confirmed this function. The mouse phenotype is reminiscent of that shown by snail mutants in Drosophila (8Go). In vitro studies have shown that Snail attenuates the cell cycle and confers resistance to cell death induced by the withdrawal of survival factors (9Go) or by DNA damage (10Go). The resistance to cell death conferred by Snail provides a selective advantage to cells to separate from the primary site and migrate.

Epithelial–mesenchymal transition (EMT) is the mechanism by which epithelial cells can dissociate from the epithelium and migrate. As such, EMT is fundamental to both normal development and the progression of epithelial tumours (11Go). Thus, Snail expression is able to trigger EMT and is increasingly recognized as an alteration in cancer. Stable expression of Snail in prototypic epithelial cell system of MDCK cells induces a complete epithelial to mesenchymal transition (5Go,6Go) and these cells overexpressing Snail exhibit tumorigenic properties when injected in nude mice (6Go). The involvement of Snail in tumour progression is also supported by its expression in invasive carcinoma cell lines (6Go), and by the expression of Snail in the invasive cells of tumours induced in the skin of mice (6Go) and in biopsies from patients with ductal breast carcinomas (12Go,13Go), gastric cancer (14Go), hepatocellular carcinomas (15Go) and synovial sarcomas (16Go). Thus, Snail expression appears to be correlated with invasive growth potential in human cancer.

In this study, to further investigate the function of Snail during cancer development, we generated mice harbouring a tetracycline-repressible Snail transgene. These mice did not exhibit morphological defects at birth, but did develop cancers similar to those associated with SNAIL expression in humans. These defects were not corrected by suppression of the Snail transgene. We found that CombitTA-Snail mouse embryonic fibroblasts (MEFs) and mice expressed Snail at levels considerably lower than those of endogenous counterparts. We further show here that CombitTA-Snail does not confer a migratory advantage, although it does induce tumour formation. CombitTA-Snail expression results in increased radioprotection in vivo. Snail expression is repressed following DNA damage in a p53-independent manner. Thus, it seems likely that failure to regulate Snail expression explains why CombitTA-Snail mice develop cancer. These results suggest that tightly graded increase of Snail can induce cancer.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Derivation of CombitTA-Snail mice
To determine the effect of upregulation of Snail expression in cancer development, we generated transgenic mice using the CombitTA system, in which the expression of Snail gene could be exogenously regulated. This system, which has the transactivator and the tet-operator minimal promoter driving the expression gene unit on a single plasmid (17Go), ensures the integration of the transactivator and reporter gene units in equal copy numbers in a direct cis-configuration at the same chromosomal locus and prevents genetic segregation of the control elements during breeding. Insertion of the mSnail gene under the control of the tetO-minimal promoter yielded the plasmid CombitTA-Snail (Fig. 1A). This was analysed in a cell system, using a murine hematopoietic precursor Ba/F3 cell line (18Go). In the absence of tetracycline, the tet-repressor protein (fused to the viral VP16 transactivator domain) binds to an engineered tet-operator minimal promoter and activates Snail transcription (CombitTA-Snail). In the presence of the tetracycline, binding is abolished and the promoter silenced (Fig. 1A). CombitTA-Snail expression was determined in transfected Ba/F3 cells after culturing for 2 days in the presence or absence of tetracycline (Fig. 1B). CombitTA-Snail was detected in Ba/F3 cells without tetracycline but not in cells cultured with tetracycline (20 ng/ml). In vitro studies have previously shown that Snail confers resistance to cell death induced by the withdrawal of survival factors (9Go). The physiological relevance of the CombitTA-Snail suppression was confirmed in vitro by assaying survival of Ba/F3 cells expressing CombitTA-Snail 24 h after IL-3 withdrawal. The effects of Snail expression on apoptosis were evaluated by analysing internucleosomal DNA cleavage leading to the formation of DNA ladders in agarose gels, which is a hallmark of apoptosis. Normally, Snail expression protects Ba/F3 cells from apoptosis following IL-3 withdrawal (Fig. 1C and D) and the level of CombitTA-Snail expression was sufficient in Ba/F3 cells to prevent cell death. The sensitivity to IL-3 removal was restored by the addition of tetracycline (Fig. 1C and D). This physiological relevance of the CombitTA-Snail function was further studied by demonstrating the nuclear localization of CombitTA-Snail protein (Fig. 2).



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  		Figure 1. CombitTA-Snail: transgene construct, expression and effect of Snail on the survival of Ba/F3 cells deprived of growth factor. (A) Schematic representation of the CombitTA-Snail vector used in this study. (B) Analysis of tetracycline-dependent Snail expression by RT–PCR in Ba/F3 cells for CombitTA-Snail (–tet, +tet in the medium). The PCR products were transferred to a nylon membrane and analysed by hybridization with a specific probe for Snail. Actin was used to check cDNA integrity and loading. (C) Survival of Ba/F3 cells expressing Snail (Ba/F3+CombitTA-Snail) in the absence of IL-3. Cells growing exponentially in IL-3 supplemented media were adjusted to 5x105 cells/ml on day 0, and cultured after removal of IL-3. The cell number of viable cells is shown for Snail-transfected Ba/F3 cells grown in the absence of IL-3 (–tet or +tet in the medium). (D) Cell death is accompanied by nucleosome laddering after IL-3 deprivation. Low-molecular-weight DNA was isolated 24 h after IL-3 deprivation from Ba/F3-CombitTA-Snail grown in the absence of IL-3 and doxycycline (–tet) (lane 1) and Ba/F3-CombitTA-Snail grown in the absence of IL-3 and with doxycycline (+tet) (lane 2). The time of treatment with doxycycline was 48 h. DNA was end-labelled, resolved by electrophoresis in a 2% agarose gel and visualized by autoradiography.

 


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  		Figure 2. Subcellular localization of CombitTA-Snail. CombitTA-Snail was transiently expressed in COS7 cells. After fixation of the cells, the localization of CombitTA-Snail protein (green) and nuclei (blue; DAPI) was examined under a fluorescent microscope. Panels before and after doxycycline treatment for Snail, nuclei alone, phase and merge images are presented (–tet or +tet in the medium). The time of treatment with doxycycline was 48 h. CombitTA-Snail was detected in COS7 cells without tetracycline but not in cells cultured with tetracycline (20 ng/ml).

 
We generated three founder transgenic lines for CombitTA-mSnail (59A, 59B and 59C) (Fig. 3A). Only two founder lines, 59A and 59B, showed germline transmission of the transgene (Table 1). In both lines, the CombitTA-Snail expression was detected in all tissues analysed (Fig. 3B). The CombitTA-Snail expression was the result of transactivation, as the suppression of expression to undetectable values was confirmed when mice were supplied with tetracycline in their drinking water (see below, Fig. 7A).



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  		Figure 3. CombitTA-Snail mice: transgene expression. (A) Identification of transgenic mice by Southern analysis of tail snip DNA after EcoRI digestion. We used the cDNA for mouse Snail for detection of the transgene. (B) Expression of the transgene was demonstrated by RT–PCR. Expression of CombitTA-Snail and endogenous Snail was analysed by RT–PCR in tissues derived of CombitTA-Snail and control mice. Actin was used to check cDNA integrity and loading.

 

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  		Table 1. Incidence and age of tumour onset in CombitTA-Snail mice
 


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  		Figure 7. Cancer development in CombiTA-Snail mice after suppression of Snail expression by tetracycline treatment. (A) Analysis of tetracycline-dependent Snail expression in peripheral blood of mice transgenic for CombitTA-Snail (–tet, +tet in water) by RT–PCR. Actin was used to check cDNA integrity and loading. (B) Representative flow cytometry phenotypic characteristics of cells from thymus, bone marrow (BM) and peripheral blood (pb) of CombitTA-Snail mice after suppression of Snail expression by tetracycline treatment (4 g/l) for 4 weeks. Cells were stained with the monoclonal antibodies and analysed by flow cytometry. The percentage of cells is indicated. (C) Representative haematoxylin/eosin stained sections of tissues in CombitTA-Snail mice after suppression of Snail expression by tetracycline treatment (4 g/l) for 4 weeks.

 
CombitTA-Snail mice show no morphological abnormalities
Cohorts of CombitTA-Snail mice were generated to analyse the effect of the Snail expression in vivo. A total of 63 transgenic animals (34 mice corresponded to line 59A and 29 mice to line 59B) were analysed in detail and similar phenotypic features were seen in both lines. CombitTA-Snail mice were born alive without overt morphological abnormalities and were fully fertile with apparently no differences in the progeny. Autopsy of pups, including extensive histological analysis, revealed no abnormality of the kidneys, skin, liver, brain, lung or gastrointestinal tract of CombitTA-Snail mice, indicating that this level of overexpression of Snail does not perturb normal embryonic development. These results contrast with the sustained expression of Snail in K14-Snail[HA] transgenic animals that results in small-sized mice and in epidermal hyperproliferation and differentiation defects in mouse skin (19Go). A priori, these differences could be simply due to variations in the Snail expression level. Although CombitTA-Snail mice show no morphological abnormalities, the thymus of CombitTA-Snail mice was small. However, T-cell numbers in peripheral blood of CombitTA-Snail mice are normal, suggesting that this overexpression of Snail does interfere with the growth of primitive mouse thymocytes but not single CD4+ and CD8+ cells. Consistent with this interpretation, analysis of thymus composition from 4-week-old CombitTA-Snail mice shows reduced cell production and differentiation towards CD4+CD8+ cells (Fig. 4). This specific T-cell differentiation block has also been observed in the thymus of Slug–/– mice (20Go).



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  		Figure 4. Deficient T-cell development in thymus of CombitTA-Snail mice. Representative analysis of the cells present in the thymus of these mice is shown. Cells isolated from a wild-type (control) and a CombitTA-Snail mouse were stained with the monoclonal antibodies and analysed by flow cytometry. The percentage of cells is indicated.

 
Cancer development in CombitTA-Snail mice
We next analysed whether the CombitTA-Snail mice develop cancer. All CombitTA-Snail mice became unwell from approximately 5–7 months of age onward (Table 1) with clinical manifestations that included decreased physical activity, tachypnea, pilo-erection, shivering and sustained weight loss, prior to sacrifice. The cancers were from both mesenchymal and epithelial origin (Table 1). The mesenchymal cancers were acute leukaemias (Fig. 5A) and lymphomas (Fig. 5B). No sarcomas were seen in any of the CombitTA-Snail mice analysed, even though with ubiquitous expression of CombitTA-Snail. Detailed analysis of the epithelial tumour cells established the diagnosis as lung carcinomas (Fig. 6A), germ cell hyperplasias (Fig. 6B) and hepatocarcinomas (Fig. 6C). We detected one type of carcinoma per animal although 20–25% of them also develop a hematopoietic neoplasia. The histological examination could not show dissemination of the carcinomas. However, histological analysis revealed marked leukaemic cell infiltration of hematopoietic and non-hematopoietic tissues. These leukaemic cells preferentially infiltrate kidney, liver and lung (Fig. 6C–E). Peripheral blood mononuclear cells from leukaemic mice were identified by flow cytometry using combination of specific antibodies. These studies defined the acute leukeamias as acute myeloid leukaemias (Fig. 5A).



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  		Figure 5. Hematopoietic cancers in CombitTA-Snail mice. (A) Phenotypic characteristics of leukaemias of CombitTA-Snail mice. Cells from bone marrow (BM), peripheral blood (pb) and spleen of CombitTA-Snail mice were analysed by flow cytometry. Cells were identified with combinations of specific antibodies. Cells (10 000) were collected for each sample and dead cells were excluded from analysis by propidium iodide staining. (B) Haematoxylin/eosin stained sections of the spleen of wild-type and CombitTA-Snail mice. The spleen from CombitTA-Snail mice shows the effacement of the normal spleen architecture. (C–E) Histologic appearance of tissues in leukaemic CombitTA-Snail mice. Leukaemic cells disobey the social order of organ boundaries and migrate as individual cells giving metastasis to different regions (liver, kidney and lung).

 


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  		Figure 6. Carcinoma development in CombitTA-Snail mice. Histological analysis of lung (A), testis (B) and liver (C) of wild-type and CombitTA-Snail mice. Representative matched tissue sections from wild-type and CombitTA-Snail mice were stained with haematoxylin/eosin. The histological sections of CombitTA-Snail lung show the presence of an adenocarcinoma (A). The histological section of CombitTA-Snail testis shows the presence of a hyperplasia of germ cells (B). The histological sections of CombitTA-Snail liver show the presence of a hepatocarcinoma (C).

 
To test the malignant potential of cells from the CombitTA-Snail mice, 1x106 peripheral blood blast cells from CombitTA-Snail leukaemias were injected subcutaneously into twelve 40-day-old nude mice. All 12 mice developed progressive tumours within 4–7 weeks of transplantation. The tumours in the nude mice were histologically identical to the original leukaemias. Overall, these data indicate that Snail is able to induce cancer development.

In vivo suppression of Snail does not block cancer development
The above results support the view that Snail expression is enough to induce cancer development. Therefore abolition of Snail overexpression might be expected to either halt or reduce the growth and/or spread of the Snail-expressing cells. To assess this, 40 leukaemic CombitTA-Snail mice were evaluated for disease progression by flow cytometry prior to and following administration of tetracycline (4 g/l in the drinking water for 2 weeks, a dose sufficient to suppress exogenous Snail expression) (Fig. 7A). None of the CombitTA-Snail mice exhibited amelioration of the leukaemic phenotype despite complete CombiTA-Snail suppression. Flow cytometry analysis identified the persistence of leukaemic cells in the peripheral blood (Fig. 7B) with infiltration of non-hematopoietic tissues evident on histology (Fig. 7C). Autopsy of these animals identified carcinomas (Fig. 7C). Thus, these results show that the alterations induced by Snail are irreversible.

A limited amount of Snail mRNA was expressed in CombitTA-Snail MEFs and mice
To understand the molecular basis underlying cancer development in CombitTA-Snail mice, we examined the expression of transgene-encoded Snail in the spleen and in primary MEFs derived from CombitTA-Snail embryos, where the endogenous Snail is expressed (Fig. 8A). The expression level of transgene-encoded Snail in spleen and MEFs of mice with respect to the endogenous expression was increased to 20% of wild-type levels (Fig. 8A). A limited amount of Snail was expressed in all tissues examined. In fact, the expression of transgene-encoded Snail was present in epithelium of CombitTA-Snail mice (Fig. 3B) and in the carcinomas appearing in CombitTA-Snail mice (Fig. 8B). Thus, these mice are an ideal in vivo model to study the consequences of low levels of Snail. In conclusion, our genetic studies point, for the first time, to the critical role for an appropriate expression level of an essential EMT regulator in cancer mouse development.



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  		Figure 8. CombitTA-Snail mice have a graded increase of CombitTA-Snail expression. (A) Quantitative real-time RT–PCR analysis of spleen and MEF RNA samples showed that CombitTA-Snail expression was increased to ~20% of endogenous Snail level in transgenic mice. CombitTA-Snail and endogenous Snail transcript numbers are shown as a percentage of ß-actin transcripts. (B) Expression of CombitTA-Snail was analysed by RT–PCR in lung carcinoma (lane 1) and hepatocarcinoma (lane 3) tissues derived of CombitTA-Snail mice. Actin was used to check cDNA integrity and loading.

 
CombitTA-Snail induces a tumorigenic but not migratory phenotype in MEFs
The above results suggested that CombitTA-Snail may not be present at a level sufficient to alter EMT in CombitTA-Snail mice. Thus, we next study the migratory/invasive properties of CombitTA-Snail MEFs. The migratory properties were analysed in a wound culture assay (6Go), where CombitTA-Snail MEFs showed a similar migratory behaviour to control MEFS. Approximately 80% of the wound surface was colonized by both control and CombitTA-Snail MEFs 15 h after the wound was made (Fig. 9A). To test the tumorigenic properties of the CombitTA-Snail MEFs, 1x106 control and CombitTA-Snail cells were injected subcutaneously into 40-day-old nude mice. Mice injected with control MEFs did not develop tumours (0 out of 10). However, CombitTA-Snail MEFs gave rise to tumours within 5–9 weeks of transplantation at the injection sites (10 out of 10) that were classified as fibrosarcomas according to histologic appearance. These results indicate that low levels of the transcription factor Snail induce a tumorigenic but not migratory phenotype in MEFs. In fact, we have not observed metastasis in CombitTA-Snail mice with carcinomas. Thus the transgene-encoded Snail may not be present at a level sufficient to alter EMT in Combi-Snail mice, what could explain why CombitTA-Snail mice show no morphological abnormalities, but this level of expression was enough to produce cancer.



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  		Figure 9. CombitTA-Snail expression in MEFs does not induce a migratory phenotype. The motility/migratory behaviour of control-MEFs (a–c) and CombitTA-Snail-MEFs (d–f) was analysed in an in vitro wound model. Confluent cultures were gently scratched with a pipette tip to produce a wound. Photographs of the cultures were taken immediately after the incision (a, d) and after 9 h (b, e) and 15 h (c, f) in culture.

 
Radioprotective potential of CombitTA-Snail mice in response to {gamma}-irradiation
The above results suggested that CombitTA-Snail expression could promote resistance to programmed cell death elicited by DNA damage. This led us to investigate the in vivo radioprotective potential of CombitTA-Snail in response to DNA damage induced by {gamma}-irradiation. CombitTA-Snail and control mice were irradiated at 950 rad (1 rad=0.01 Gy). As shown in Figure 10A, CombitTA-Snail mice survive longer than control mice. These results indicate that CombitTA-Snail expression results in increased radioprotection.



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  		Figure 10. Effect of irradiation on survival of CombitTA-Snail mice. (A) CombitTA-Snail (30 animals) and control mice (30 animals) were irradiated at 950 rad to determine their survival after DNA damage. The radiation dose was given as a split dose of equal intensity, 4 h apart. (B) Levels of p53 protein in CombitTA-Snail and control BM cells after {gamma}-irradiation. p53 protein was detected by western blotting. Actin was used as a loading control. The time points are in hours.

 
Exposure to ionizing radiation causes an increase in the intracellular levels of p53 (21Go) and in vitro studies have also shown that aberrant overexpression of Snail and Slug alters the response to genotoxic stress by increasing the level of p53 (10Go). Thus, we next explored if the radioprotective potential of CombitTA-Snail was based on interference with p53 activation. As shown in Figure 10B, we measured the p53 protein levels at different time points in bone marrow cells derived from both CombitTA-Snail and control mice after DNA damage induced by {gamma}-irradiation. The activation of p53 in both control and CombitTA-Snail cells was similar (Fig. 10B), indicating that p53 regulation in response to DNA damage is not affected in CombitTA-Snail cells.

DNA damage regulates Snail mRNA expression
The above results suggested that Snail expression protects cells from DNA damage. This led us to investigate whether DNA damage regulates Snail expression. As a model for in vitro studies to determine whether Snail has a functional role in response to DNA damage-mediated cellular activities, we used MEFs (Fig. 11A). We treated MEFs of different genotypes with the chemotherapeutic agent, doxorubicin, which causes DNA damage. The expression of the p53 target gene p21 was used as a positive control. As shown in Figure 11A, DNA damage inhibits expression of Snail in MEFs in a p53-independent manner. To confirm this result, approximately 4935 bp of the promoter region of the human SNAIL gene was cloned upstream of a luciferase reporter gene (pGL3-basic). To directly assess the ability of P53 to activate transcription from DNA sequences present in the SNAIL promoter, an expression vector containing a human P53 cDNA (22Go) was co-transfected into U2OS cells along with the reporter vector containing the SNAIL promoter. Co-expression of P53 did not result in an increase in luciferase activity compared to the activity with the empty vector (Fig. 11B). These results further indicate that P53 does not regulate the SNAIL promoter.



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  		Figure 11. Identification of Snail as a DNA damage transcriptionally regulated gene. (A) Northern blot analysis of Snail expression in MEFs from different genotypes following DNA damage. RNAs were prepared from cells treated/not treated with doxorubicin (±dox). After hybridization with a Snail cDNA probe, the same blot was rehybridized with BclxL and p21 probes as positive controls. Loading was monitored with ARPP-PO. (B) P53 does not transactivate the SNAIL promoter. Luciferase reporter assays demonstrate independent responsiveness of the human SNAIL reporter to P53. This promoter was activated in response to agents that induce Snail transcription, as the addition of the phorbol ester PMA (33Go). The number shown at the left of the reporter constructs denotes the 5'-boundaries (bp upstream of the initiation site). (C) In vivo regulation of Snail expression in response to DNA damage. In spleen of mice 6 h after 5 Gy of {gamma}-radiation, Snail expression is reduced in both the wild-type and the p53–/– spleen tissues. Northern blots were hybridized with Snail and ARPP-PO (U, untreated).

 
Next, we examined the p53-independent regulation of Snail expression following DNA damage in vivo. We treated wild-type and p53–/– mice with 5 Gy of {gamma}-radiation and analysed the expression of Snail in spleens by northern blot (Fig. 11C). Six hours after irradiation, the expression of Snail was down-regulated in both control and p53–/– mice. Therefore, we conclude that Snail expression is similarly modulated in vivo following DNA damage. Overall, our results demonstrate the requirement of a critical level of Snail for cancer development and it seems likely that failure to regulate Snail explains why CombitTA-Snail mice develop cancer.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
During the last years, different studies indicated the involvement of Snail in mesenchymal phenotype in cancer (5Go,6Go,11Go–16Go). Nevertheless, the molecular mechanisms by which Snail participates in these biological processes are not yet clear. We have utilized the single-plasmid system containing the regulating and expression elements of the original binary tetracycline system to allow induction and tight control of gene expression by tetracycline in mice (17Go) to try to understand the relevance of Snail to human cancer development. In vitro studies have shown that Snail confers resistance to cell death induced by the withdrawal of survival factors (9Go). The physiological relevance of the CombitTA-Snail suppression was confirmed in vitro by assaying survival of Ba/F3 cells expressing CombitTA-Snail after IL-3 withdrawal. The analysis of the Snail-expressing mice identified that these mice develop cancer, mainly hematopoietic tumours. It is believed that the resistance to cell death conferred by Snail provides a selective advantage to cell migration important to cancer development (9Go). Thus, the hematopoietic cancers observed in the CombitTA-Snail mice represent an in vivo demonstration of the idea that transformation depends upon genetic changes that allow undifferentiated cells to grow outside their normal environment (23Go). Thus, these results provide evidence that Snail expression facilitates cell migration. However, the survival conferred by Snail, while reversible in vitro (Fig. 1), can escape such control in vivo.

In the mouse, the Snail gene has been shown to trigger EMT, an important pathway to acquisition of the invasive phenotype in epithelial solid tumours (5Go,6Go). Our data did not support this observation, with both no-epithelial alterations and non-invasive carcinoma development in CombitTA-Snail mice. However, CombitTA-Snail mice expressed a limited amount of Snail, although level sufficient to promote resistance to cell death elicited by growth factor withdrawal (Fig. 1). Thus, the transgene-encoded Snail may not be present at a level sufficient to alter EMT in our mice, but this level of expression was enough to induce cancer. It appears that Snail must be kept above a certain threshold level to achieve normal development. Consistent with this interpretation, CombitTA-Snail induced a tumorigenic but not migratory phenotype in MEFs. These findings indicate Snail does not require tumour formation before dissemination can place. However, these results cannot exclude a role for Snail in carcinoma development in a context where epithelial cells show or accumulate previous tumour alterations.

Slug expression confers resistance to programmed cell death, a function shared by Snail (9Go), elicited either by growth factor (20Go) or by DNA damage (10Go,24Go,25Go). In this sense, Slug has been shown to play similar roles to Snail in several systems (12Go,13Go,26Go,27Go), and, thus, other members of the Snail family of transcription factors could also been involved in similar biological functions to those described herein to Snail. Stable expression of Slug in prototypic epithelial cell system of MDCK cells also induce a complete epithelial to mesenchymal transition and show similar migratory ability to that of MDCK-Snail cells (28Go). But it is not clear whether this functional equivalence also occurs during tumour progression, as Slug is expressed in different carcinoma-derived cell lines regardless of their phenotype in terms of invasiveness (6Go). Recently our group demonstrated that Slug is required for cancer development in mice (29Go). However, we did not detect an increase in Slug expression associated to overexpression of Snail in CombitTA-Snail mice. Similarly, Slug does not influence the expression of Snail in MDCK cells (28Go).

Our results show that ‘increased’ Snail expression induces cancer in mice with high frequency. These results suggested that Snail expression was protecting cells from death by genetic alterations as a consequence of an inherent, basal level of genetic instability (23Go). In fact, CombitTA-Snail expression resulted in increased radioprotection. Thus, constitutive activation of Snail could confer radioresistance properties to the tumour-target cells. In concert with these results, we show that both in vivo and in vitro Snail expressions are modulated in response to DNA damage. However, although in vitro studies have suggested that aberrant expression of Snail alters the response to genotoxic stress by increasing p53 levels (10Go), p53 response to DNA damage was not affected in CombitTA-Snail mice. Our results connect DNA damage with the requirement of a critical level of an EMT regulator for cancer development and it seems likely that failure to regulate Snail explains why CombitTA-Snail mice develop cancer. These findings further indicate that overexpression of Snail by human tumours could be of importance to cell fate selection by genotoxic anticancer agents. Further studies will be needed to determine if Snail attenuates stress-induced apoptosis response.

Does the Snail–DNA damage interaction contribute to a physiological defence mechanism exploited by human cancers? Snail is able to trigger EMT, an important pathway to acquisition of the invasive phenotype in epithelial solid tumours. Thus, under physiological conditions, DNA damage decreases Snail expression and could contribute to a transient inhibition of migratory capacity of tumour-target cell. With constitutive expression of Snail during transformation, this control is lost. Thus human cancers that overexpress Snail may have a survival advantage to genotoxic and potentially other forms of stress by exploiting physiological mechanisms that evolved for the EMT, raising the possibility of strategies based on Snail for the treatment of human cancer.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Generation of transgenic mice and treatments
The cDNA for mouse Snail was cloned into the Combi-tTA vector (17Go). Linear DNA fragments for microinjection were obtained by NotI digestion and injected into CBAxC57BL/6J-fertilized eggs. We identified transgenic mice by Southern analysis of tail snip DNA after EcoRI digestion as described (30Go). We used the cDNA for mouse Snail for detection of the transgene. Founder mice were crossed to the C57BL6 mice for five generations to establish co-isogenic transgenic mice. Similar phenotypic features were seen in all assays for both the CombitTA-Snail transgenic lines generated. Mice aged 5–6 weeks were irradiated using a caesium source and maintained in microisolator cages on sterilized food and acidified sterile water.

Histological analysis
Mice included in this study were subjected to standard necropsy. All major organs were closely examined under the dissecting microscope, and samples of each organ were processed into paraffin, sectioned and examined histologically. All tissue samples were taken from homogenous and viable portions of the resected sample by the pathologist and fixed within 2–5 min of excision. Haematoxylin- and eosin-stained sections of each tissue were reviewed by a single pathologist (T.F.). For comparative studies, age-matched mice were used (wild-type or Combi-Slug mice in the continuous presence of tetracycline).

Cell culture
Cell lines used include Ba/F3 cells (18Go) and COS7 cells. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). When required, 10% WEHI-3B-conditioned medium was added as a source of IL-3.

Subcellular localization of CombitTA-Snail
CombitTA-Snail was transiently expressed in COS7 cells. After fixation of the cells, immunostaining was carried out by using anti-Snail antibody (173EC3F9) (Franci et al. in preparation)—as described (31Go). The localization of the CombiTA-Snail protein was examined under a fluorescent microscope.

Cell transfection and cell survival assay
Ba/F3 cells were transfected by electroporation (960 µF, 220 V) with 20 µg of either CombitTA-Snail. The neomycin-resistant pool of cells (Ba/F3+CombitTA-Snail) were analysed by RT–PCR for CombitTA-Snail expression in the presence and in the absence of tetracycline (20 ng/ml). These cells were resistant to IL-3 withdrawal when grow in the absence of tetracycline. Cells were screened for resistance to IL-3 withdrawal and cell viability was determined by Trypan blue exclusion.

Culture of MEFs
Heterozygous p53+/– (Jackson Laboratories) and p21+/– (a gift of M. Serrano) mice were crossed to obtain wild-type and null p53–/– and p21–/– embryos, respectively. Primary embryonic fibroblasts were harvested from 13.5 d.p.c. embryos. Head and organs of day 13.5 embryos were dissected; fetal tissue was rinsed in PBS, minced and rinsed twice in PBS. Fetal tissue was treated with trypsin/EDTA and incubated for 30 min at 37°C and subsequently dissociated in medium. After removal of large tissue clamps, the remaining cells were plated out in a 175-cm2 flask. After 48 h, confluent cultures were frozen down. These cells were considered as being passage one MEFs. For continuous culturing, MEF cultures were split into 1:3. MEFs were grown at 37°C in DMEM (Boehringer Ingelheim) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Boehringer Ingelheim). All the cells were negative for mycoplasma (MycoAlertTM Mycoplasma Detection Kit, Cambrex).

DNA damage experiments
Cells were plated at 106 cells per 10 cm dish, and the day after, they were treated with 0.2 µg/ml of doxorubicin (Sigma) (32Go). After 12 h, cells were collected for RNA preparation.

Low-molecular-weight DNA analysis
Low-molecular-weight DNA was isolated as follows. Cells were collected into 1.5 ml of culture medium and microcentrifuged for 1 min at 1500 rpm (400g), and the pellet was suspended in 300 µl of proteinase K buffer. After overnight incubation at 55°C, DNA was ethanol-precipitated, suspended in 200 µl of TE buffer containing 50 µg/ml of RNase A, and incubated at 37°C for 2 h. DNA was extracted with phenol and chloroform, and precipitated with ethanol. Aliquots of DNA (2 µg) were end-labelled with {alpha}32-dCTP and electrophoresed on 2% agarose gels. After electrophoresis, the gel was blotted onto Hybond-N (Amersham) and autoradiographed for 2 h at –70°C.

Reverse transcription–PCR (RT–PCR) and real-time PCR quantification
To analyse expression of CombitTA-Snail and endogenous Snail in mouse cell lines and mice, RT was performed according to the manufacturer's protocol in a 20-µl reaction containing 50 ng of random hexamers, 3 µg of total RNA and 200 U of Superscript II RNase H– reverse transcriptase (GIBCO/BRL). The sequences of the specific primers were as follows: Combi-polyA-B1: 5'-TTGAGTGCATTCTAGTTGTG-3'; mSnailF: 5'-CAGCTGGCCAGGCTCTCGGT-3'; mSnailB: 5'-GCGAGGGCCTCCGGAGCA-3'. Endogenous Snail expression was analysed with mSnailF and mSnailB primers and exogenous Snail expression with mSnailF and Combi-polyA-B1 primers. Amplification of ß-actin RNA served as a control to assess the quality of each RNA sample. The PCR conditions used to amplify CombitTA-Snail and endogenous Snail were as follows: 94°C for 1 min, 56°C for 1 min and 72°C for 2 min for 40 cycles for CombitTA-Snail and 30 cycles for endogenous Snail, respectively. The PCR products were confirmed by hybridization with specific internal probes. Real-time quantitative PCR was carried out for the quantitation of both CombitTA-Snail and endogenous Snail. Fluorogenic PCRs were set up in a reaction volume of 50 µl using the TaqMan PCR Core Reagent kit (PE Biosystems). cDNA amplifications were carried out using the same primers in a 96-well reaction plate format in a PE Applied Biosystems 5700 Sequence Detector. Thermal cycling was initiated with a first denaturation step of 10 min at 95°C. The subsequent thermal profile was 40 cycles of 95°C for 15 s, 56°C for 30 s and 72°C for 1 min. Multiple negative water blanks were tested and a calibration curve was determined in parallel with each analysis. The ß-actin endogenous control (PE Biosystems) was included to relate both CombitTA-Snail and endogenous Snail to total cDNA in each sample.

Phenotype analysis
The following anti-mouse monoclonal antibodies from Pharmingen were used for cytometry staining: CD45R/B220 (B-cell-specific), IgM (B-cell-specific), Mac1 (myeloid-cell-specific), Gr-1 (myeloid-cell-specific), CD4 (T-cell-specific) and CD8 (T-cell-specific). Single cell suspensions from different tissue samples obtained by routine techniques were incubated with purified anti-mouse CD32/CD16 (Pharmingen) to block binding via Fc receptors and with an appropriate dilution of the different antibodies at room temperature or 4°C. The samples were washed twice with PBS and resuspended in PBS. Dead cells in samples were excluded by propidium iodide staining. The samples and the data were analysed in a FACScan using CellQuest software (Becton Dickinson).

Tumorigenicity assay
To test the tumorigenicity of the various CombitTA-Snail cancers and MEFs, 4–6-week-old athymic (nude) male mice were injected subcutaneously on both flanks with 106 cells resuspended in 200 µl of phosphate-buffered saline (PBS). The animals were examined for tumour formation every week.

Luciferase assays
Approximately 4935-bp upstream promoter sequence of SNAIL was isolated from a P1 clone containing the SNAIL gene (Genome Systems) and cloned into the luciferase reporter plasmid pGL3-basic (Promega) and termed PSNAIL-4935. For reporter assays, U2OS cells were transfected using Dual-Luciferase (Promega) with normalization to Renilla luciferase, and mean±standard error was determined from at least three data points. U2OS cells were maintained in DMEM supplemented with 10% FBS. When indicated, cells were supplemented with phorbol 12-myristate 13-acetate (PMA) (from Sigma).

Northern blot analysis
Total cytoplasmic RNA of different MEFs and spleen tissues from both untreated and 5 Gy-irradiated wild-type and the p53–/– mice was glyoxylated and fractionated in 1.4% agarose gels in 10 mM Na2HPO4 buffer (pH 7.0). After electrophoresis, the gel was blotted onto Hybond-N (Amersham), UV-cross-linked and hybridized to 32P-labelled mouse Slug cDNA probe. Loading was monitored by reprobing the filter with a ARPP-P0 probe.

Western blot analysis
Bone marrow cells were collected by flushing the marrow cavity of femurs. Western blot assays were done using extracts from 1x107 BM cells per lane. Extracts were normalized for protein content by Bradford analysis (Bio-Rad Laboratories, Inc., Melville, NY, USA) and Coommasie blue gel staining. Lysates were run on a 10% SDS–PAGE gel and transferred to a PVDF membrane. After blocking, the membrane was probed with the following primary antibodies: mouse p53 was detected using the antibody FL-393 (Santa Cruz) and the polyclonal antibody C-11 (Santa Cruz) was used to detect actin. Reactive bands were detected with an ECL system (Amersham).

Migration assays
The migratory/motility behaviour of transfectant cells was analysed by the wound assay. Monolayers of confluent cultures were lightly scratched with a Gilson pipette tip and, after washing to remove detached cells, the cultures were observed at timely intervals as previously described (6Go).


   ACKNOWLEDGEMENTS
 
We thank all members of lab 13 for their helpful comments and constructive discussions on the manuscript. We are grateful to Dr Pedro Soria for continuous and generous help with the mice irradiation, to Dr Tyler Jacks for various useful reagents, to Dr Manuel Serrano for the p21 mice, to Dr Martin Haas for the human p53 expression vector and to Dr H. Bluethmann for the Combi-tTA vector. This work was supported by MEyC (BIO2000-0453-P4-02, SAF2003-01103, FIT-010000-2004-157 and PETRI no. 95-0913.OP), Junta de Castilla y León (CSI03A05), ADE de Castilla y León (04/04/SA/0001), FIS (PI020138, G03/179 and G03/136), Fundación de Investigación MMA and USAL-CIBASA project. M.S.M. was supported by FIS grant no. PI041271 and M.P.C. is a recipient of a MCyT fellowship.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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