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Cell, Tumor, and Stem Cell Biology |
1 Breast and Gynecological Cancer Group, Centro Nacional de Investigaciones Oncológicas; 2 Departamento de Bioquímica, UAM, Instituto de Investigaciones Biomédicas "Alberto Sols," CSIC-UAM, Madrid, Spain; and 3 Institut d'Investigació Bellvitge, IDIBELL, Barcelona, Spain
Requests for reprints: Amparo Cano, Instituto de Investigaciones Biomedicas, CSIC-UAM, Arturo Duperier, 4. 28029 Madrid, Spain. Phone: 34-91-5854411; E-mail: acano{at}iib.uam.es. Jose Palacios, Servicio de Anatomia Patologica, Hospital Virgen del Rocio, Avenida Manuel Siurot, 43013 Sevilla, Spain. Phone: 34-95-5013029; E-mail: jose.palacios.sspa{at}juntadeandalucia.es.
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Introduction |
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An extensive genetic program must be set in motion for this phenotypic change to take place, altering the expression of an extensive array of molecules. However, the possibility exists that similar but distinct genetic programs of EMT might be at play in specific cell contexts, and that these may involve different aspects of epithelial plasticity (5, 8). Recent advances in our understanding of the regulation of EMT have benefited significantly from studies of development and tumor progression (2, 9). Indeed, recent studies on the molecular mechanisms underlying E-cadherin down-regulation in carcinomas, an event essential for EMT and tumor invasion, have proved to be particularly informative. Several transcriptional repressors of E-cadherin have recently been isolated, including the zinc finger factors Snail (10, 11) and Slug (12, 13), the two-handed zinc factors SIP-1 (ZEB-2) and 
EF1 (ZEB-1; refs. 14, 15), and the bHLH factors E12/E47 and Twist (16, 17). The Snail, Slug, and E47 repressors all induce a similar phenotype when overexpressed in epithelial MDCK cells, eliciting complete EMT at both the morphologic and behavioral levels (11, 12, 16). The expression of Snail, Slug, and E47 in carcinoma cell lines with invasive and metastatic properties (11, 12, 16) and the expression of Snail in undifferentiated breast carcinomas and invasive hepatocarcinomas (18, 19) are evidence that these genes are also involved in tumor progression (reviewed in ref. 7). Moreover, it has recently been suggested that Snail is involved in the local recurrence of breast carcinomas (20). Furthermore, the differential expression of Snail, Slug, SIP1, and/or Twist has been observed in distinct types of gastric carcinomas (21) as well as in ovarian and breast, prostate, or esophageal squamous cell carcinomas (22–24).
The aim of the present study was to obtain additional insights into the role of different E-cadherin repressors in EMT. To this end, we analyzed the gene expression profile of the well-characterized MDCK-Snail, MDCK-Slug, and MDCK-E47 cell lines (11, 12, 16, 25) compared with control MDCK-CMV cells by cDNA microarrays and RNA arbitrarily primed-PCR (RAP-PCR). In this way, we were able to identify the existence of common and specific gene targets regulated by the expression of the three factors in the same genetic background. Validation of several selected markers in vitro and in xenografts supports the differential participation of the three repressors in EMT and tumor progression.
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Materials and Methods |
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Differential Expression by RAP-PCR
For RAP-PCR assays, total RNA from the different cell lines was extracted from 5 x 106 cells growing at low or high density using the isolation kit RNeasy Mini (Qiagen GmbH, Hilden, Germany). Fifty nanograms of RNA were reverse transcribed using an oligo-dT primer and MLV reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD); 2.5 µL of the reverse-transcribed mRNAs were used as templates in PCR reactions [1 unit of Taq polymerase (Boehringer Mannheim, Mannheim, Germany), 50 µmol/L of each deoxynucleotide triphosphate, and 1 µCi [
-32P]dATP, 3.7 mmol/L MgCl2] done with the arbitrary primers over 35 low-stringency cycles (94°C 30 seconds, 42°C 45 seconds, 72°C 1.5 minutes). Fingerprints were resolved on 6% denaturing acrylamide gels at 55 W for 3.5 hours or until the xylene cyanol front had reached the bottom of the gel. Gels were dried and the amplified products were visualized by autoradiography. All PCR reactions were done at least twice and the results were similar in all cases. The sequences of the primers used for reverse transcription-PCR (RT-PCR) and RAP-PCR are indicated in Supplementary Table S1. All combinations of primers used in this assay gave reproducible fingerprints that contained at least 50 bands. Fingerprints were compared and the differences in the intensity of the bands were evaluated from the scanned films using Phoretix 1D software (Newcastle-upon-Tyne, United Kingdom). Selected bands were cut out and identified by automated DNA sequencing after cloning into the pCR2.1 vector (Invitrogen, Inc., Carlsbad, CA). The sequences obtained were matched with those included in the GenBank using the BLAST software.4 The origin of the bands was assessed by hybridization to the RAP-PCR gel after transfer to a blotting membrane (Qiabrane Nylon Plus, Qiagen, Chatsworth, CA). Hybridization was done at 65°C under high-stringency conditions to a probe that was generated from the cloned band and labeled with -32P by random primer method.
Differential Expression Profile Analysis
RNA extraction, amplification, labeling, microarray hybridization, and analysis. Total RNA from the different cell lines was extracted from duplicate 80% confluent cultures using the TRIZOL reagent following the instructions of the manufacturers (Life Technologies). Three micrograms of total RNA were used to synthesize the double-stranded cDNA as previously described (26) and 3 µg of amplified RNA were used to generate fluorescence antisense RNAs using the SuperScript enzyme protocol (Life Technologies).
The Oncochip microarray platform v1.1 contains 9,726 clones corresponding to 6,386 different genes, and it includes 2,489 duplicate clones that enable the reproducibility to be assessed. Duplicate samples from each of the MDCK transfectants were labeled with dUTP-Cy5 and hybridized against the dUTP-Cy3-labeled MDCK-CMV controls as previously described (26). Two additional hybridizations using the reciprocal fluorochrome labeling were done; thus, a total of four hybridizations were done for each condition. The slides were washed, dried, and then scanned in a ScanArray 5000 XL scanner (GSI Lumonics, Kanata, Ontario, Canada) to obtain 10-µm resolution images. These were then quantified using the GenePix Pro 6.0 program (Axon Instruments, Inc., Union City, CA). The array data were included in a GEO public repository (GSE4151).
The fluorescence intensity measurements of each array experiment were processed using the GenePix Pro 6.0 and MS Excel programs as described (26). For statistical analysis, we selected genes of which the expression differed by a factor of at least 2 with respect to the MDCK-CMV control. A hierarchical clustering method was applied to group the genes and samples on the basis of the similarities in expression, and the unsupervised analyses were visualized using the SOTA and TreeView software assuming euclidean distances between genes.5 Genes with potentially significant changes in expression between the three transcription factors were identified using the POMELO program.6 We tested the null hypothesis of equal means among the three groups by ANOVA, computing P values using a permutation test. To select differentially expressed genes, we adjusted for multiple testing using the false discovery rate method (27). Here we report differential expression in genes with a false discovery rate–adjusted P < 0.05.
Validation Studies
Northern blotting. To analyze the levels of gene expression, 15 to 30 µg of total RNA isolated by TRIZOL from the cell lines grown to 80% confluence were separated on 1% formaldehyde agarose gels, blotted onto nylon membranes (Schleicher & Schuell Bioscience, Keene, NH) by the capillary method, and UV cross-linked. The integrity and loading of RNA samples were assessed by methylene blue staining of the transferred RNA. The origin of the probes and specific hybridization conditions are described in Supplementary Methods.
For Northern blot analysis of RAP-PCR bands, RNA was prepared from either low-density or high-density cultures, as indicated, and the membranes were hybridized at high-stringency conditions using [-32P]cDNA from the cloned bands as probes.
RT-PCR analysis. Two micrograms of total RNA were used in RT-PCR experiments using specific primers for transcript detection of MITF (all isoforms): direct, 5'-GTGCAGACCCACCTGGAAAAC-3', and reverse, 5'-AGTTAAGAGTGAGCATAGCCATAG-3'. Amplification was done over 40 cycles (94°C 30 seconds, 55°C 30 seconds, 72°C 2 minutes). RT-PCR for canine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to control for the amount of cDNA and these amplifications were done as previously described (16).
In addition, to study mRNA of mouse and human Snail, Slug, and E47, we did a RT-PCR using as primers mouse E47 (forward, 5'-GTACAGATGAGGTGCTGTCCCT-3'; reverse, 5'-GACACCTTCTCCTCCTCCCTC-3'), human Slug (forward, 5'-CGCTCCTTCCTGGTCAAGA-3'; reverse, 5'-TTGCGTCACTCAGTGTGC-3'), and human E47 (forward, 5'-GAGGAGAAAGACCTGAGGGACC-3'; reverse, 5'-ACCTGACACCTTTTCCTCTTCTC-3'). Primer sequences used for mouse Snail, mouse Slug, human Snail, and mouse and human GAPDH have previously been described (11, 12). PCR products were obtained after 30 cycles of amplification with an annealing temperature of 60°C to 65°C.
Immunofluorescence and Western blot analysis. For immunofluorescence, cells grown on coverslips were fixed in either methanol (–20°C) or 3.7% formaldehyde (for 30 minutes at room temperature) and then incubated with the primary and secondary antibodies as described elsewhere (11, 12, 28). For Western blot analysis, total cell extracts from the different cell lines were obtained in radioimmunoprecipitation assay buffer and analyzed as previously described (11, 16, 28). The list of primary and secondary antibodies used is provided in Supplementary Methods. Blots were developed using the enhanced chemiluminiscence detection system (Amersham Pharmacia Biotech, Uppsala, Sweden).
Promoter analyses. The activity of PAI-1 (29) and cyclin D1 (30) promoters was done by detecting promoter driven luciferase activity as previously described for the E-cadherin promoter (12). The indicated cell lines were transfected with 200 ng of the reporter constructs and 20 ng of a TK-renilla construct as a control of transfection efficiency. Extracts were obtained 24 hours after transfection and luciferase and renilla activities were evaluated with a dual-luciferase reporter kit (Invitrogen). The activities were normalized to those of control MDCK-CMV cells and the experiments were done on duplicate samples at least twice for each experimental condition. Values represent the mean ± SD.
Induction of Xenografted Tumors and Immunohistochemistry
Xenografted tumors from the MDCK-Snail, MDCK-Slug, and MDCK-E47 cell lines were obtained following s.c. injection into 8-week-old female BALB/c nude mice as previously described (2 x 106 cells per flank: refs. 16, 25). Growing tumors in mice were measured every 2 days using calipers by determination of the two orthogonal external diameters and mice were killed when tumors reach a size of 0.5 cm3. Tumors were surgically excised and processed for histology and immunohistochemistry. Mice were housed and maintained under specific pathogen-free conditions and used in accordance with institutional guidelines and approved by the Use Committee for Animal Care. A total of 8 to 12 tumors from each cell line were generated and at least four different tumors derived from each cell line were analyzed.
Immunohistochemical staining was done using the Envision method (Dako, Glostrup, Denmark) with a heat-induced antigen retrieval step where the sections were immersed in boiling 10 mmol/L sodium citrate (pH 6.5) for 2 minutes in a pressure cooker. Mouse anti-human antibodies E-cadherin (4A2C7 clone, Zymed, San Francisco, CA), N-cadherin (3B9 clone, Zymed), p120 (98 clone, BD Biosciences, San Jose, CA), and SPARC (15G12 clone, Novocastra, Newcastle, United Kingdom) were applied at 1:200, 1:25, 1:500, and 1:50 dilutions, respectively, and the rabbit anti-human antibody ID3 (Santa Cruz Biotechnology, Santa Cruz, CA) was applied at 1:50 dilution. The distribution of the different markers was scored as membranous, cytoplasmic, and/or nuclear depending on the marker analyzed.
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Of the total number of candidate genes, 12 were down-regulated and 20 were up-regulated by expression of either of the factors (Fig. 1D), indicating the pleiotropic effects of the three transcriptional factors as repressors and activators of common primary or secondary targets. However, there were differences in the level of induction or repression of any selected gene by these factors, as witnessed for the SPARC (Table 2 ). Furthermore, the remaining genes showed a different expression pattern depending on the factor expressed. Indeed, some genes were down-regulated by one factor and up-regulated by the other two, or they were only regulated by one or two of the factors (Fig. 1D; Supplementary Table S2). Therefore, to better establish which genes might be differentially and specifically regulated by each transcription factor, we did a supervised analysis using the POMELO tool and ANOVA. Using this supervised approach, 100 genes were identified as differentially expressed with a significant association between the factors (false discovery rate–adjusted P < 0.05; Table 2 and Supplementary Table S1, asterisks). These genes also showed relatively homogeneous expression between the replicates of each transcription factor when compared with the variation between the lines (see Fig. 1A). Together, the data provided by the cDNA array analysis suggest that the three transcription factors, Snail, Slug, and E47, induce common and specific gene expression programs that lead to EMT as well as conferring specific functional properties.
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3, and integrin ß6 subunits) are down-regulated, whereas some mesenchymal markers (SPARC, collagen type I, III, and V subunits, and integrin 5), protease-related activities (PAI-1 and TIMP1), and actin cytoskeleton associated proteins (moesin, WASPIP, and caldesmon) are up-regulated (Tables 2 and 3
). Interestingly, some of these EMT-related genes were also detected in the RAP-PCR analysis in Snail- and E7-expressing cells (i.e., up-regulated SPARC; Table 1).
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-catenin, was also validated in Western blots (Fig. 3A).
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5 integrin, vinculin, and caldesmon was validated at the protein level by immunofluorescence and/or Western blotting (Figs. 2 and 3A). In Northern blots, the up-regulation of collagen 15A1 and cadherin 6 transcripts was validated in the three cell types (Fig. 3C). The microarray data indicating that collagen 3A1 was much more strongly induced in Snail- and Slug-expressing cells (24.6- and 14.3-fold, respectively) than in E47 cells (2.6-fold induction) were also confirmed in Northern blots (Fig. 3C). In contrast, the analysis of CTGF expression by Northern blot showed a strong expression in Snail cells, much lower levels in Slug cells, and its apparent absence in E47 cells (Fig. 3D), evidence that CTGF transcripts might be differentially expressed in MDCK-Snail cells. Importantly, a similar behavior in the expression of these various EMT markers was detected in several independent clones generated from each of the transfectant cell type and showing similar levels of the corresponding transgene (Supplementary Fig. S3A and B). Finally, the differential up-regulation of the protease-related activity PAI-1 by the three factors (strong in Slug and Snail cells and moderate in E47 cells) was validated by immunofluorescence (Fig. 3F) and promoter analysis. Indeed, the relative levels of PAI-1 promoter activity in the three cell lines closely paralleled the microarray data (Fig. 3E, compare to PAI-1 data in Table 2).
Therefore, these results indicate that Snail, Slug, and E47 regulate the expression of common and specific pathways that may contribute to general and particular aspects of the EMT. They also indicate that the three factors can act both as repressors and activators of primary or secondary target genes required for EMT.
Transcription-related genes. About 8% of the genes identified are directly or indirectly related to transcriptional process (Fig. 1C; Table 2). Interestingly, only two of these seem to be regulated in a common fashion by Snail, Slug, and E47. The microphthalmia-associated transcription factor (MITF; ref. 34) was down-regulated by all three genes and the transcription factor 4 (TCF4; a bHLH factor also known as E2-2; ref. 35) was up-regulated in the three cell lines, although to a different extent (Tables 2 and 3). The expression of six transcription related genes was regulated in Slug and E47 cells, and these included members of the bHLH family (BHLHB2 and TWIST) of the Id subfamily (ID3 and ID4) and of the leucine zipper family (MAFG and KRML; Table 2). Interestingly, eight genes were only up-regulated in Slug cells, including several coactivators and modifiers of chromatin (CITED2, AIB3, and SMARCC1), proliferation response factors (SRF and Sp2), and zinc finger factors, whereas one gene only seemed to be up-regulated in E47 cells (HDAC3; Table 1). The differential up-regulation of ID3, ID4, Twist, and KRML mRNAs in E47 and Slug cells was validated in Northern blots, where Twist and ID3 transcripts also seemed to be up-regulated, but to a lower extent, in Snail cells (Fig. 4A ; Supplementary Fig. S3). Because of the relevance of the Id factors in tumor progression (36), we analyzed by Northern blot the expression of another two members of the Id family (ID1 and ID2) that showed nonconfident results in the microarrays. Both ID1 and ID2 transcripts were strongly up-regulated in Slug- and E47-expressing cells (Fig. 4A, top two rows; Supplementary Fig. S3) and ID1 mRNA was also induced in MDCK-Snail cells, albeit to a lesser extent. The differential regulation of ID1 and ID3 transcripts was also confirmed in independent clones from each transfectant cell type (Supplementary Fig. S3C). The alterations in MITF expression observed in the microarrays were validated by RT-PCR confirming that transcripts common to all described isoforms of MITF (34) were repressed in Snail-, Slug-, and E47-expressing cells, the stronger repression being detected in the latter cell line (Fig. 4B).
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To gain further information about TGF-ß-regulated genes, the expression of TGF-ß1 in Snail, Slug, and E47 cells was analyzed in Northern blots. TGF-ß1 transcripts were up-regulated in each of the cell lines but with quantitative differences (Slug > Snail > E47; Fig. 4C), supporting the differential regulation of TGF-ß-mediated signaling pathways by the three factors. On the other hand, two cyclin-dependent kinase inhibitors (p21Cip and p18) were up-regulated in Slug and E47 but no significant changes were observed in Snail-expressing cells. Promoter analysis of cyclin D1 confirmed the microarray data for Slug- and E47-expressing cells and the expression of this gene was not significantly altered in Snail-expressing cells (Fig. 4E). A similar increase in the amounts of p21Cip protein was detected in the three cell lines when compared with control cells (Fig. 4F), indicating that additional posttranscriptional mechanisms influence p21Cip expression.
Genes related to other functions. The expression of genes that are involved in apoptosis was also altered, mainly in Slug- and E47-expressing cells. These included signaling and regulatory proteins of several apoptotic pathways, such as the up-regulation of GAS1 and MAZ or the down-regulation of CFLAR (Supplementary Table S1). In addition, a few angiogenesis-related genes were seen to be differentially expressed in the three MDCK cell lines (Supplementary Table S1; Table 3). Whereas Jagged 1 was up-regulated in each line, and particularly in Slug cells (18-fold induction), VEGF was up-regulated in Slug and E47 cells. The expression of other genes was only affected in Slug-expressing cells (i.e., EPAS1, endoglin, or endothelin). The increase in VEGF expression in Slug and E47 cells was validated in Northern and Western blots (Fig. 4D).
Finally, an important fraction of the genes identified were associated to metabolism and/or basic cellular functions (Fig. 1C), some common to the three cell types and others specific (Table 3; Supplementary Table S1). This further indicates that the regulation of metabolism might be an important function of these transcription factors, as recently suggested for Snail (37).
Histopathology and expression of EMT markers in xenografted tumor samples. To confirm the biological relevance of the identified gene targets and in particular of EMT-related genes, xenografted tumors induced by MDCK-Snail, MDCK-E47, and MDCK-Slug cells were subjected to histologic and immunohistochemical analysis. According to previous studies (25), tumors induced by MDCK-Snail and MDCK-E47 cells were highly undifferentiated spindle cell tumors without evidence of epithelial differentiation (Fig. 5, left and right ). In contrast, tumors induced by MDCK-Slug cells grew slowly7 and showed areas of glandular differentiation surrounded by proliferating malignant undifferentiated spindle cells (Fig. 5, middle). This phenotype is consistent with a diagnosis of carcinosarcoma. Thus, MDCK-Slug xenografts provided an interesting model for this tumor type, as well as provided evidence of the reversibility of the EMT phenotype.
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EF1/Zeb2) seem to fulfill this requirement, and they induce complete EMT when overexpressed in epithelial cell systems (10–12, 15–17). In addition, they have been functionally or causally implicated in developmental EMT processes, and their patterns of expression in carcinoma cell lines and tumors are fully compatible with their participation in tumor progression (7, 9). Nevertheless, it is still unclear whether different EMT programs, corresponding to particular states of epithelial plasticity, might be induced by different signals or factors (5, 8, 39). Moreover, it remains unclear whether the various inducers of EMT identified play a specific or redundant role in tumorigenesis. To gain additional insight into the implication of Snail, Slug, and E47 in EMT and tumor progression, we analyzed the gene expression profile induced by each independent factor on the same genetic background. As such, we took advantage of the previously characterized MDCK-Snail, MDCK-Slug, and MDCK-E47 cell lines stably expressing each of the transcription factors (11, 12, 16, 25), and compared gene expression in these cells with control mock-transfected MDCK cells using RAP-PCR and cDNA microarrays. Significantly, only 32 of the 228 (14%) candidate genes identified in the microarray analysis were regulated in a similar fashion (up- or down-regulated) by the three factors, whereas most of the genes were found to be regulated by different combinations of two of the factors or simply by just one of them. Therefore, these results indicate that Snail, Slug, and E47 might be involved in the regulation of common and specific pathways that contribute to general and particular aspects of EMT, as well as to other biological functions.
Unsupervised hierarchical clustering of the candidate genes showed that the genetic profile of MDCK-Slug and MDCK-E47 cells was more alike than that of MDCK-Snail cells. In principle, this was quite surprising because Snail and Slug are highly homologous members of the evolutionary conserved Snail gene family, sharing highly conserved DNA binding and transactivation domains (40). Indeed, these two genes are capable of mediating related developmental EMT events in distinct species (9). However, there are several lines of evidence indicating that these two members of the Snail family may play distinct roles, at least in mammals. For instance, whereas Snail is essential for early mouse development, being necessary for gastrulation to proceed correctly (41), Slug is dispensable during murine embryonic development but it is necessary in the adult for the differentiation and/or maintenance of specific cell lineages (42, 43). In addition, both factors have distinct patterns of expression in embryonic regions of EMT and/or in established mesenchymal tissues (9, 11, 44). The microarray data presented here fully support the differential implication of Snail and Slug in the regulation of specific gene programs, which, although sharing some common targets, are quite divergent. Thus, it seems likely that each factor is probably fulfilling specific functions in EMT and promoting distinct responses to external signals. In this context, the differential expression of some cell cycle mediators and effectors of apoptosis observed in Snail- and Slug-expressing cells might well be related to the different in vivo growth potentials observed for MDCK-Snail and MDCK-Slug xenografts.7 The specific molecular mechanisms responsible for the differential target regulation by Snail and Slug are currently unknown. The different affinities for specific E-boxes in gene promoters might account for the differential regulation of some genes, as shown for E-cadherin (12). We can also speculate that the recruitment of specific corepressors and coregulators by either factor could also be relevant (45). The studies necessary to clarify these important issues are presently under way.
In contrast, the strong similarity in the expression profiles of MDCK-Slug and MDCK-E47 cells, sharing 30% of the candidate genes, was somewhat unexpected. This uniformity might indicate that both factors converge on common primary or secondary target genes, thus regulating similar biological processes. Notably, the majority of genes common to Slug- and E47-expressing cells were up-regulated (49 of 59), suggesting that they may share common transcriptional regulators. Indeed, detailed inspection of the 18 transcription related genes shows that 6 of them are jointly up-regulated in MDCK-Slug and MDCK-E47 cells, including members of the HLH family and the leucine zipper family. The specific involvement of some of these transcription factors in the genetic programs elicited by Slug and E47 factors is presently under investigation. It will be particularly interesting to further understand the relationship and/or hierarchy between Slug/E47 and the Twist factors in specific stages of tumor progression like intravasation (17). In addition, the strong up-regulation of ID1 and ID2 transcripts observed in MDCK-Slug and MDCK-E47 cells, and of ID3 in the three cell types (including MDCK-Snail cells), contrasted sharply with the down-regulation of ID2 (and ID3) reported in EMT induced by TGF-ß signals in other cell systems (46). Up-regulation of ID3 mRNA by Snail, Slug, or E47 expression in MDCK cells was also confirmed at the protein level in the xenografts induced by the three cell lines. However, the striking differences in the global gene expression programs detected between MDCK-Snail and MDCK-E47 cells, particularly with respect to transcription and cell signaling genes, might explain the differential invasive and angiogenic behavior of both cell types in vivo (25). Together, these results provide evidence for the existence of distinct genetic programs underlying the EMT mediated by different signaling pathways and/or factors, in full agreement with the proposal of epithelial plasticity (5, 8, 39).
Remarkably, among the EMT-related genes detected in our microarray analysis, 16 (36% of EMT genes) were regulated in a common fashion by the expression of either Snail, Slug, or E47, whereas the rest seemed to be specifically regulated by only one or two of the factors. Analysis of the common regulated genes showed that 6 were down-regulated and 10 were significantly up-regulated (see Table 3). Importantly, most of them were validated in several independent clones from each transfectant cell type. It seems logical that these sets of genes should therefore participate in the acquisition of general functions common to the EMT process, such as the disruption of epithelial polarity and phenotype (E-cadherin and keratin 19), mesenchymal cell-extracellular matrix adhesion properties (collagen subunits I2 and III1, integrin 5, and SPARC), remodeling of the extracellular matrix (PAI-1 and TIMP1), and dynamic actin cytoskeletal organization (caldesmon). In fact, some of these common genes have been functionally implicated in or associated with EMT (3, 5, 8, 9). Interestingly, some of these genes have recently been identified as targets of Snail or Slug in other cell systems: SPARC and TIMP1 in melanocytes (47) or cytokeratin 19 in breast cells (48). The other EMT-related genes that were seen to be differentially regulated by only one or two of the factors (like CTGF as a potential target of Snail) may contribute to specific aspects of the process that may only be required in some specific biological situations. Indeed, these genes may confer particular migratory and/or metastatic properties to tumor cells, as suggested for CTGF in bone metastasis of breast carcinomas (49). These important issues should be clarified in further studies. In this context, it also interesting to note that our analysis revealed few similarities with the genes regulated after transient expression of Snail in colon carcinoma cells (37). However, the important differences in the experimental design of both studies, including induced versus stable expression of Snail, absence of complete EMT in the colon system, and use of different microarray platforms, preclude a direct comparison.
Immunohistochemical analysis of the xenograft tumors generated by the MDCK transfectants confirmed the in vivo expression of related EMT markers. Indeed, this approach showed that the expression of Snail and E47 produced spindle cell tumors, whereas the expression of Slug induced tumors with mixed carcinomatous and sarcomatous components, consistent with the diagnoses of carcinosarcoma. These results further support the differential implication of Snail and Slug factors and also indicate the reversibility of the EMT phenotypes induced, supporting the previous proposal (3, 7) that EMT is a dynamic and transient process during tumor progression. Analysis of the expression levels of each factor in tumor biopsies should contribute to clarify the specific participation of the factors in tumor progression, something that awaits the availability of specific and reliable antibodies to each factor.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Amalia Montes, Vanesa Santos, and Raquel Marcos for their excellent technical collaboration; Centro Nacional de Investigaciones Oncológicas Biotechnology Units (Immunohistochemistry, Comparative Pathology and Confocal Microscopy); and our colleagues who provided the reagents.
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Footnotes |
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G. Moreno-Bueno and E. Cubillo contributed equally to this work. G. Moreno-Bueno is a junior investigator of the Ramón y Cajal Program (2004).
Current address for Victoria Bolós: Centro Nacional de Biotecnología, Madrid, Spain.
4 http://www.ncbi.nlm.nih.gov/blast. 
5 http://bioinfo.cnio.es/cgi-bin/tools/clustering/sotarray.
6 http://www.genoma.wi.mit.edu/MPR/software.
7 V. Bolós and A. Cano, unpublished data.
Received 2/ 9/06. Revised 6/ 2/06. Accepted 7/26/06.
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 S.-H. Park, L. W. T. Cheung, A. S. T. Wong, and P. C. K. Leung Estrogen Regulates Snail and Slug in the Down-Regulation of E-Cadherin and Induces Metastatic Potential of Ovarian Cancer Cells through Estrogen Receptor {alpha} Mol. Endocrinol., September 1, 2008; 22(9): 2085 - 2098. [Abstract] [Full Text] [PDF]
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H. Peinado, G. Moreno-Bueno, D. Hardisson, E. Perez-Gomez, V. Santos, M. Mendiola, J. I. de Diego, M. Nistal, M. Quintanilla, F. Portillo, et al. Lysyl Oxidase-Like 2 as a New Poor Prognosis Marker of Squamous Cell Carcinomas Cancer Res., June 15, 2008; 68(12): 4541 - 4550. [Abstract] [Full Text] [PDF]
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D. Sarrio, S. M. Rodriguez-Pinilla, D. Hardisson, A. Cano, G. Moreno-Bueno, and J. Palacios Epithelial-Mesenchymal Transition in Breast Cancer Relates to the Basal-like Phenotype Cancer Res., February 15, 2008; 68(4): 989 - 997. [Abstract] [Full Text] [PDF]
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D. Olmeda, G. Moreno-Bueno, J. M. Flores, A. Fabra, F. Portillo, and A. Cano SNAI1 Is Required for Tumor Growth and Lymph Node Metastasis of Human Breast Carcinoma MDA-MB-231 Cells Cancer Res., December 15, 2007; 67(24): 11721 - 11731. [Abstract] [Full Text] [PDF]
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K. M. Newkirk, A. E. Parent, S. L. Fossey, C. Choi, H. L. Chandler, P. J. Rajala-Schultz, and D. F. Kusewitt Snai2 Expression Enhances Ultraviolet Radiation-Induced Skin Carcinogenesis Am. J. Pathol., November 1, 2007; 171(5): 1629 - 1639. [Abstract] [Full Text] [PDF]
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 J. Mejlvang, M. Kriajevska, C. Vandewalle, T. Chernova, A. E. Sayan, G. Berx, J. K. Mellon, and E. Tulchinsky Direct Repression of Cyclin D1 by SIP1 Attenuates Cell Cycle Progression in Cells Undergoing an Epithelial Mesenchymal Transition Mol. Biol. Cell, November 1, 2007; 18(11): 4615 - 4624. [Abstract] [Full Text] [PDF]
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R. G. Hardy, C. Vicente-Duenas, I. Gonzalez-Herrero, C. Anderson, T. Flores, S. Hughes, C. Tselepis, J. A. Ross, and I. Sanchez-Garcia Snail Family Transcription Factors Are Implicated in Thyroid Carcinogenesis Am. J. Pathol., September 1, 2007; 171(3): 1037 - 1046. [Abstract] [Full Text] [PDF]
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T. Shirakihara, M. Saitoh, and K. Miyazono Differential Regulation of Epithelial and Mesenchymal Markers by {delta}EF1 Proteins in Epithelial Mesenchymal Transition Induced by TGF-beta Mol. Biol. Cell, September 1, 2007; 18(9): 3533 - 3544. [Abstract] [Full Text] [PDF]
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X. Gu, L. F. Zerbini, H. H. Otu, M. Bhasin, Q. Yang, M. G. Joseph, F. Grall, T. Onatunde, R. G. Correa, and T. A. Libermann Reduced PDEF Expression Increases Invasion and Expression of Mesenchymal Genes in Prostate Cancer Cells Cancer Res., May 1, 2007; 67(9): 4219 - 4226. [Abstract] [Full Text] [PDF]
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