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Human Molecular Genetics Advance Access originally published online on October 3, 2005
Human Molecular Genetics 2005 14(22):3361-3370; doi:10.1093/hmg/ddi366
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

E-cadherin and vitamin D receptor regulation by SNAIL and ZEB1 in colon cancer: clinicopathological correlations

Cristina Peña1,{dagger}, José Miguel García1,{dagger}, Javier Silva1, Vanesa García1, Rufo Rodríguez5, Isabel Alonso2, Isabel Millán3, Clara Salas4, Antonio García de Herreros6, Alberto Muñoz7 and Félix Bonilla1,*

1Department of Medical Oncology, 2Department of Surgery, 3Biostatistics Unit and 4Department of Pathology, Hospital Universitario Puerta de Hierro, Madrid, Spain, 5Department of Pathology, Hospital Virgen de la Salud, Toledo, Spain, Hospital Universitario Puerta de Hierro, Madrid, Spain, 6Unitat de Biología Cellular i Molecular, Institut Municipal d'Investigació Mèdica-Universitat Pompeu-Fabra, Barcelona, Spain and 7Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Spain

* To whom correspondence should be addressed at: Department of Medical Oncology, Hospital Universitario Puerta de Hierro, C/ San Martín de Porres, 4, 28035 Madrid, Spain. Email: felixbv{at}stnet.es

Received September 8, 2005; Accepted September 23, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
E-cadherin (CDH1) gene expression is strictly regulated. The transcriptional factors SNAIL and ZEB1 are involved in its repression, whereas activation of vitamin D receptor (VDR) by vitamin D induces its transcription. We study the expression and functional correlation of SNAIL, CDH1, VDR and ZEB1 genes and examine their possible involvement in colon cancer. The expression of these four genes was measured by real time-PCR in 114 patients with colorectal cancer, and tumor characteristics were analyzed in each patient. SNAIL expression was associated with downregulation of CDH1 (P<0.001) and VDR (P<0.001) gene products. We also found a positive correlation between CDH1 and VDR expressions. However, the association between SNAIL and CDH1 was not found in patients with high expression of ZEB1. We observed a correlation between downregulation of: a) ZEB1 and presence of polyps in surgical resections; b) VDR and poor differentiation and c) CDH1 and poor differentiation, vascular invasion, presence of lymph node metastases and advanced stages; as well as a trend toward a correlation between SNAIL expression in tumors and vascular invasion. The correlations between SNAIL, CDH1, VDR and ZEB1 and the association between reduced expression of CDH1 and VDR and aggressive tumor characteristics emphasize the value of analyzing these genes in colon cancer patients for prognostic purposes and for predicting response to possible therapies with vitamin D or its analogs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
E-cadherin, the prototype and best-characterized member of the cadherin family, is located in adherens junctions. It contributes to the maintenance of the adhesive and polarized phenotype of epithelial cells where it is mainly expressed (1Go). When E-cadherin is downregulated, the epithelial cells acquire a fibroblastoid morphotype, accompanied by acquisition of invasive and migratory properties (2Go–4Go). This event is critical for invasion and metastasis of carcinoma cells (5Go,6Go). Therefore, the loss of E-cadherin expression can be considered as an indicator of poor clinical prognosis.

Therefore, it is important to identify the molecular mechanism that regulates the expression of E-cadherin. Mutations (7Go–9Go), loss of heterozygosity (LOH) (10Go) or promoter hypermethylation (11Go,12Go) are the mechanisms reported to inactivate CDH1 gene expression. In addition, E-cadherin is regulated by transcriptional factors such as SNAIL, which represses expression by binding to 5'-CACCTG-3' sequences of the promoter region (13Go).

The Snail family of zinc-finger proteins is required to complete gastrulation and the migration of the neural crest from the neural tube in avian and murine embryos (14Go,15Go). In these processes, SNAIL represses E-cadherin expression, causing epithelial cells to acquire fibroblastic properties and increasing cell migration (16Go). It is also reported that SNAIL confers resistance to cell death (17Go), which provides a selective advantage for tumors that become malignant.

In contrast, the active metabolite of vitamin D, 1{alpha},25-dihydroxyvitamin D3, induces the expression of E-cadherin gene in cells expressing the nuclear vitamin D receptor (VDR) (18Go). Vitamin D and its analogs regulate gene expression by binding to VDR, which is a ligand-modulated transcription factor (19Go). High VDR expression is associated with favorable prognosis in colorectal cancer cell lines. Therefore, downregulation of this receptor may be involved in pathogenesis and be a predictive marker for this malignancy (20Go). In this regard, we have recently reported a correlation between the overexpression of SNAIL and the loss of VDR in vitro and in a small number of colorectal cancers (21Go).

E-cadherin and VDR can be also regulated by ZEB1 transcriptional factor. This factor differently modulates both genes, as it has been reported to repress E-cadherin transcription (22Go,23Go) and stimulate VDR protein levels (24Go).

ZEB1 and its homolog ZEB2 are two-handed zinc finger transcription factors that present identical overall gene structure and significant sequence similarity (25Go,26Go). Both recognize the same E-boxes that are bound by SNAIL (25Go,26Go). It is thus conceivable that they compete for the same binding sites in their target genes. Furthermore, ZEB1 either represses or activates specific target genes depending on the differential recruitment of co-repressors or co-activators (27Go). ZEB1 can bind to the p300 and P/CAF co-activators, displacing the CtBP co-repressor and activating target genes such as some TGFß-dependent genes (27Go). Therefore, recruitment of p300 and P/CAF with displacement of CtBP by ZEB1 may be responsible for the transcriptional activation of VDR (24Go). Similar activities have also been described for ZEB1 and ZEB2, for example, in the regulation of the E-cadherin gene, which is repressed by both factors (22Go,28Go,29Go).

Here we studied the associations between the expression of SNAIL, CDH1, VDR and ZEB1 genes in a large series of colorectal cancer patients. There was a high correlation between the overexpression of SNAIL and the loss of expression of CDH1 and VDR. However, the correlation between CDH1 and SNAIL was not found when ZEB1 was overexpressed. Significant associations were found between reduced CDH1 expression and pathological characteristics such as vascular invasion, presence of lymph node metastasis and poor tumor differentiation, between the downregulation of VDR and tumor de-differentiation and also between the downregulation of ZEB1 and the presence of polyps in the surgical resections.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Descriptive statistics of clinicopathological data
The series analyzed comprised 63 men (55.3%) and 51 women (44.7%), age range 37–89, median 71±11. Among the 114 patients, 61 (53.5%) presented tumor in the left colon, 33 (28.9%) in the right colon and 20 (17.5%) in the rectum. Differentiation was good in 72 (63.2%), moderate in 35 (30.7%) and poor in 7 (6.1%). Forty-three (37.7%) patients did not present lymph node metastases, whereas 71 (62.3%) had at least one node affected. Nine (7.9%) were stage I, 62 (54.4%) stage II, 40 (35.1%) stage III and 3 (2.6%) stage IV. Forty-five (39.5%) patients had vascular invasion. Thirty-one (27.2%) patients presented polyps in the surgical resections.

CDH1, VDR, ZEB1 and SNAIL expression data
The expression levels of CDH1, VDR, SNAIL and ZEB-1 RNA in a series of 114 colorectal tumors and their normal mucosa counterparts were measured by real time (RT)-PCR as described in Material and Methods. The medians of CDH1, VDR and ZEB1 expression were 0.96, 2.11 and 2.30, respectively. The minimum and the maximum values of CDH1, VDR and ZEB1 expression were 0.01 and 146.00, 0.06 and 131.80 and 0.04 and 42.78, respectively. CDH1, VDR and ZEB1 expression quartiles 25 and 75% were 0.38 and 2.53, 0.86 and 5.05 and 0.47 and 8.17, respectively. SNAIL mRNA expression was detected in 72 of 114 patients (63.2%).

To validate these results and examine whether E-cadherin protein expression in tumors cells was homogeneous, we performed immunohistochemical analyses. Tumors were clearly classified into E-cadherin positive and E-cadherin negative when compared with normal mucosa (Fig. 1). We did not detect significant changes in E-cadherin expression between different regions (central area and invasive front) from the same tumor (Fig. 1). Importantly, there was a strict correlation between the results of E-cadherin RNA and protein expression: immunostaining was negative in those showing RNA downregulation by RT-PCR.



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  		Figure 1. Immunohistochemical analysis of E-cadherin expression. (A) Normal colon mucosa. (B) Normal mucosa (right) and tumor (left) of a patient who was positive for E-cadherin. (C and D) Central area and invasion front, respectively, of an E-cadherin-positive tumor. (E and F) central area and invasion front, respectively, of an E-cadherin-negative tumor.

 
Deregulation of SNAIL and ZEB1 in tumor cells
We analyzed the repressors of E-cadherin expression in this series of tumors. As antibodies against these proteins are not available, the study was performed by RT-PCR. First we validated this method to rule out the possibility that the expression of SNAIL and ZEB-1 could correspond to contaminating stromal cells rather than carcinoma cells in the biopsies. With this objective, tumor samples were processed in order to separate epithelial and non-epithelial cell populations, using antibody-coated beads, as described in Materials and Methods. Non-epithelial cytokeratin-negative cells constituted 5–15% of the whole tumor sample disaggregates, but were absent in epithelial-enriched fractions following separation with paramagnetic beads (Fig. 2). Emphasizing the validity of the procedure, only 0–3% of cells which did not bind to the paramagnetic beads were epithelial cytokeratin-positive cells (Fig. 2).



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  		Figure 2. Images under the fluorescence microscope of the different cellular fractions. (A) Whole disaggregated with some non-epithelial cells (DAPI positive and CK negatives) (arrow) and epithelial cells (DAPI and CK positives). None of the non-epithelial cells was joined to the magnetic beads. (B) Epithelial-enriched fraction where all the cells observed were epithelial cells. (C) Supernatant fraction where epithelial cells were not observed.

 
High levels of SNAIL expression were observed in six out of 10 samples of total tumor disaggregates in which geometric average of SNAIL expression was 0.21. SNAIL overexpression was found in the epithelial fraction (retained in the beads) (geometric average=0.18) but not in the supernatant (non-epithelial) fraction (geometric average=0.83) (Fig. 3). Moreover, when SNAIL was not overexpressed in the whole biopsy (four out of 10, geometric average 1.37), it was detected neither in the epithelial fraction (geometric average=1.22) nor in the supernatant fraction (geometric average=1.21) (Fig. 3).



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  		Figure 3. Geometric average and standard error of SNAIL expression of different cell populations isolated from disaggregated tumor specimens.

 
In the same way, low levels of ZEB1 were found in four out of the 10 samples (40%, geometric average=8.37). All of these four samples showed ZEB1 downregulation only in the epithelial-enriched fraction (geometric average=13.61) but not in the non-epithelial fraction (geometric average=2.1) (Fig. 4). In whole disaggregated cell populations with normal ZEB1 RNA expression levels (six out 10, geometric average=1.23), similar low levels were found in epithelial (geometric average=2.54) and non-epithelial fractions (geometric average=0.84) (Fig. 4). No ZEB1 overexpression was found in this series.



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  		Figure 4. Geometric average and standard error of ZEB1 expression of different cell populations isolated from disaggregated tumor specimens.

 
Therefore, these results strongly suggest the expression of SNAIL detected in colorectal tumor samples corresponded to tumor tissue and not to contaminating non-epithelial stromal cells.

Correlations between SNAIL, VDR, CDH1 and ZEB1 expressions
We found a high inverse correlation between the expression of SNAIL and that of VDR and CDH1 genes. When SNAIL was expressed in tumors, CDH1 and VDR were downregulated, whereas in the absence of SNAIL, CDH1 and VDR RNA levels were similar or higher than those in normal mucosa (Table 1) (Fig. 5).


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  		Table 1. Correlations between expression levels of SNAIL, VDR, CDH1 and ZEB1
 


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  		Figure 5. (A) Quantitative expression of CDH1 and VDR RNA, log(N/T), in the colon cancer series with regard to SNAIL expression. (B) Box-plot of CDH1 and VDR expression and their statistical significance (ANOVA) regarding SNAIL expression in the colorectal cancer series. The graphs show the quartiles 25, 50 and 75, values lower than 1.5 box lengths and the outliers (O) of the CDH1 and VDR expression.

 
We found a strict correlation between VDR and CDH1 expressions. In 38 tumors with low expression of CDH1, expression of VDR was low in 25 (66%) and high in only two (P<0.001). Likewise, in 38 tumors with high expression of E-cadherin, expression of VDR was high in 25 (66%) and low in only two (P<0.001). In contrast, there was no correlation between the levels of expression of ZEB1 and those of the other genes studied. ZEB1, CDH1 and VDR expressions were analyzed in relation to that of SNAIL. A trend toward statistical association between ZEB1 expression and CDH1 downregulation was observed in patients without SNAIL expression (P=0.070) (Table 2). In addition, no significant correlation was found between ZEB1 and CDH1 expressions in patients expressing SNAIL (P=0.610) (Table 2), and no association between ZEB1 and VDR was found independent of SNAIL expression.


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  		Table 2. Cross-tabs between CDH1 and ZEB1 tertiles in patients without SNAIL expression (P=0.070) and in patients with SNAIL presence (P=0.610)
 
Also, we analyzed the association of SNAIL expression with that of VDR or CDH1 in relation to ZEB1 expression. Interestingly, at high levels (in the highest tertile) of ZEB1, the geometric average of CDH1 gene downregulation in tumor tissue (relative to that in normal tissue) was independent of SNAIL expression. Thus, the average of CDH1 expression was 1.51 when SNAIL was expressed and 0.89 when SNAIL was absent (P=0.457) (Fig. 6). However, at normal levels of ZEB1 (second tertile) or in the absence (lowest tertile) of ZEB1 expression, the inverse correlation between CDH1 and SNAIL was clear (P=0.006 and P<0.001, respectively) (Fig. 6). Thus, in tumors with high expression of ZEB1, no correlation was found between overexpression of SNAIL and downregulation of CDH1, as there were many cases of downregulation of CDH1 in the absence of SNAIL expression. In contrast, the association between overexpression of SNAIL and downregulation of VDR was independent of the level of ZEB1 expression.



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  		Figure 6. Box-plot: relationships between CDH1 expression levels and the presence or absence of SNAIL regarding ZEB1 expression. Levels of ZEB1 have been distributed in tertiles: (A) Low ZEB1 expression. (B) Normal ZEB1 expression. (C) High ZEB1 expression. The graphs show the quartiles 25, 50 and 75, values lower than 1.5 box lengths and the outliers (O) of CDH1 expression. P-values were calculated by ANOVA test.

 
VDR, CDH1, SNAIL and ZEB1 expressions and pathological data
We found a significant correlation between low levels of expression of VDR and poor tumor differentiation. Among 114 patients 72 (63.2%) presented well-differentiated tumors with a VDR downregulation geometric average of 1.65. Thirty five (30.7%) had moderate tumor differentiation and a geometric average of 2.85 and 7 (6.1%) with poor tumor differentiation showed a geometric average of 6.97 (P=0.008).

Analysis of the relationship between CDH1 expression and the pathological data revealed some significant associations. CDH1 downregulation was significantly correlated with vascular invasion, presence of lymph node metastases and poor tumor differentiation. In addition, a trend toward significance was found at advanced stages, which reached statistical significance when stage IV patients were not considered. This is a consequence of the small number of stage IV patients, a common characteristic of colorectal cancer series (Table 3).


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  		Table 3. Associations between the expression of E-cadherin gene and clinicopathological characteristics
 
There was no significant association between SNAIL presence and any of the parameters analyzed. However, analysis of SNAIL expression with vascular invasion showed a trend toward a correlation (P=0.07) (data not shown). In contrast, there was a significant association between downregulation of ZEB1 and the presence of polyps in the surgical specimen (P=0.041) (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this work, we report a significant correlation between the expression of SNAIL and the downregulation of VDR and CDH1 genes in a large series of 114 patients with colorectal cancer. Unexpectedly, the correlation between the expression of SNAIL and the downregulation of CDH1 was lost when ZEB1 was overexpressed. These results coincide with previous findings in cultured cells and in a small series of colorectal carcinomas, which showed correlation between the overexpression of SNAIL and the loss of VDR and CDH1 gene expression (21Go). CDH1 transcription can be repressed by different transcriptional factors, such as SNAIL, ZEB1 or ZEB2, which bind to the specific E-boxes in the E-cadherin promoter (13Go,22Go,28Go,29Go). Downregulation of VDR by SNAIL is also accounted for by the binding of this repressor to the VDR promoter in colon carcinoma cells (21Go). In another study, Rosivatz et al. (30Go) reported similar results in diffuse-type gastric carcinomas.

SNAIL and ZEB1 genes are expressed by mesenchymal cells (13Go,16Go,25Go) and, presumably, by stromal cells. In our tumor samples, up to 25% cells were non-epithelial. In this context, the results on SNAIL or ZEB1 deregulation observed in tumor tissue might be influenced by the expression of these genes in non-epithelial cells, masking the expression in epithelial tumor cells. This possibility has, however, been ruled out, as similar changes to those observed in the tumors were detected using an epithelial-enriched cell populations. We thus conclude that alterations found in the expression of these genes in the whole tumor samples of the 114 patients studied correspond to carcinoma cells.

A trend toward statistical significance was observed between ZEB1 upregulation and CDH1 downregulation in patients without SNAIL expression. Moreover, the correlation between the overexpression of SNAIL and the downregulation of CDH1 was not found in the patients in whom ZEB1 was overexpressed. As ZEB1 can downregulate E-cadherin in several cell lines (22Go,23Go,28Go,29Go), the levels of co-regulators of ZEB1, or a covalent modification (27Go), may modulate the activity of this transcriptional factor in tumor patients. On the basis of these observations, we hypothesize a model in which the presence of SNAIL as well as of the levels of different ZEB1 co-factors might modulate ZEB1 activity, thus preventing its inhibitory effect on the CDH1 promoter. In the absence of SNAIL, the repressive action of ZEB1 is unmasked. Likewise, the lack of correlation between SNAIL and CDH1 may thus be explained when ZEB1 is overexpressed, as this gene product would repress E-cadherin independently of SNAIL. Putatively, the deregulation of different CDH1 modulators might mask its repression by SNAIL, which may explain why the correlation between SNAIL and CDH1 is lower than that observed between SNAIL and VDR. More studies in larger series of patients and analyses at the molecular level should be performed to test these hypotheses.

CDH1 is considered an invasion-suppressor gene and its loss is an indicator of high tumor aggressiveness (5Go,31Go–33Go). Previous immunohistochemical studies have shown a reduction of E-cadherin protein in colon cancer (11Go,34Go). Our data confirm this finding and reveal a significant association between the loss of E-cadherin and pathological data considered as factors of poor prognosis, such as vascular invasion, presence of lymph node metastases, advanced stages and poor tumor differentiation.

Some authors have reported differences in the pattern of E-cadherin immunostaining between the central areas of primary carcinomas and the invasion front of the tumor (35Go,36Go). This observation may correspond with the loss of cellular adhesion associated with invasion (5Go,6Go), but it is not known whether this mechanism is ubiquitous in the tumor periphery. In our series of colon carcinomas, no significant intra-tumor differences were found in E-cadherin staining. This finding might be influenced by the low proportion of poorly differentiated tumors (6%) in the series. In any case, all of the E-cadherin protein-negative (or negative immunostained) tumors showed low mRNA expression levels. Thus, these results indicate that the quantification of CDH1 RNA in the whole biopsy is a good marker of E-cadherin expression.

We also found a correlation between downregulation of VDR and poor differentiation of the tumors and a trend toward statistical significance between overexpression of SNAIL and vascular invasion. In addition, the presence of polyps in the surgical specimen of colon cancer resection correlated with ZEB1 downregulation and might indicate a greater focal proliferation of the cells. If so, the formation of polyps could be considered as a first step in tumor growth and de-differentiation. It is also possible that ZEB1 depletion in tumors might enhance cell growth. The loss of VDR and ZEB1, and probably the presence of SNAIL, could thus be considered as predictors of poor clinical prognosis in colon cancer.

The antitumoral properties of non-hypercalcemic vitamin D3 analogs have been described both in vitro and in vivo (18Go,37Go–40Go). These compounds require VDR for their biological activity (18Go,41Go). Moreover, epidemiological and preclinical data indicate that vitamin D may reduce the risk of several types of cancer (42Go,43Go). Vitamin D or its analogs could be used to treat cancer: for example, in prostate cancer patients, treatment with 1,25-dihydroxyvitamin D3 slows the rate of increase of prostate specific antigen (44Go). However, other studies found little or no antitumoral response in patients treated with these compounds (45Go–48Go), possibly because the patients studied presented high levels of SNAIL and consequently a loss of VDR or simply because of constitutive alterations in VDR integrity or expression. It has recently been reported that calcium supplementation reduces the risk of colorectal adenoma recurrence when vitamin D (25-hydroxyvitamin D3) serum levels are higher than the median of the studied series (49Go).

Our study indicates that the balance between SNAIL, VDR and ZEB1 expressions sets the level of CDH1 transcription and determines the characteristics of tumors and possibly their behavior. The results emphasize the value of analyzing these genes in colon cancer patients in order to predict their clinical outcome. They may also be of consequence for treatment; as responsiveness to vitamin D analogs requires the integrity of VDR, analysis of its expression could be crucial in guiding selection of those colon cancer patients likely to respond to a possible innocuous treatment with vitamin D analogs. This selection would be similar to that carried out in breast cancer patients selected for hormonotherapy on the basis of the presence or absence of estrogen receptors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Patients, tumor samples and RNA extraction
The present study, approved by the Research Ethics Board of our hospital, was based on a consecutive series of 114 patients undergoing surgery for colorectal cancer between January 1998 and January 2003, all of whom gave written informed consent. All patients were considered sporadic cases because no clinical antecedents of FAP were reported and those with clinical criteria of hereditary non-polyposis colorectal cancer (Amsterdam criteria) were excluded. Both normal and tumor tissues were obtained sequentially immediately after surgery, immersed in RNA laterTM (Ambion Inc, Austin, TX, USA), snap-frozen in liquid nitrogen and stored at –80°C in our laboratory until processing.

A series of 10 patients operated for colorectal cancer were used for the selection of epithelial cells. Both normal and tumor samples were obtained after surgery and processed immediately as described below.

All tumors were histologically examined by a pathologist to: a) confirm the diagnosis of adenocarcinoma, b) verify the presence of tumor and select those samples with at least 75% of tumor tissue and c) establish the pathological stage.

For the 114 patients, total RNA was extracted from tumor and normal samples using RNeasy Mini Kit (Quiagen Inc., Hilden, Germany). The RNA extracted was quantified spectrophotometrically.

RT-PCR
SNAIL mRNA was not detected in normal tissues; therefore, SNAIL expression was evaluated only as presence or absence in tumor tissues. A SNAIL retrogene, SNA1P, localized at chromosome 2q34-X, shows high homology to SNAIL (50Go). Expression of SNA1P in epithelial cell lines induces epithelial–mesenchymal transitions and represses CDH1 expression, although less strongly than SNAIL (50Go). In order to perform the specific amplification of SNAIL, primers were designed from a region with 16 bp differences between SNAIL and SNA1P. Amplification of SNA1P was thus avoided, as checked by sequencing of the PCR fragments obtained.

VDR, ZEB1 and CDH1 mRNA levels were calculated in the normal and tumor counterpart samples in a relative quantification approach where the amount of the targets was expressed in relation to the geometric average of the three reference housekeeping genes: TATA binding protein (TBP), succinate dehydrogenase complex subunit A (SDHA) and ubiquitin C (UBC), as described elsewhere (51Go). The relative concentrations of target and reference genes were calculated by interpolation using a standard curve of each gene generated with a serial dilution of a cDNA prepared from RNA extracted from SW480-ADH cells for CDH1, VDR and ZEB1 genes. The expression level of a target gene in a patient was calculated as the ratio of its expression in normal to that in tumor tissues (N/T). The quantitative mRNA analysis was confirmed by independent analysis. The primers used are shown in Table 4. For the synthesis of the first strand of cDNA, 400 ng of total RNA was retro-transcribed using the Gold RNA PCR Core Kit (PE Biosystems) following the manufacturer's instructions. Random hexamers were used as primers for cDNA synthesis.


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  		Table 4. Sequence of the primers and conditions for the amplification of each gene
 
Real-time PCR was performed in a Light-Cycler apparatus (Roche Diagnostics, Mannheim, Germany) using the LightCycler-FastStart DNA Master SYBR Green I Kit (Roche Diagnostics). Each reaction was performed in a final volume of 20 µl containing 2 µl of the cDNA product sample, a different MgCl2 concentration for each primer (Table 4), 0.5 µM of each primer and 1x reaction mixture including FastStar DNA polymerase, reaction buffer, dNTPs and SYBR green. Thermal cycling for all genes was initiated with a denaturing step at 95°C for 10 min and followed by 40 cycles [denaturing at 94°C 0 s, annealing at a different temperature for each gene for 5 s, (Table 4), and elongation at 72°C for 5 s, in which fluorescence was acquired]. At the end of the PCR cycles, melting curve analyses were performed, as well as electrophoresis of the products on non-denaturing 8% polyacrylamide gels, followed by sequencing, in order to validate the generation of the specific PCR product expected.

Immunohistochemistry
Immunophenotypic analysis of several samples taken from various parts of the same tumor section from each of 25 patients was performed according to standard procedures, with overnight incubation in the presence of anti-E-cadherin DAKO antibody. Immunodetection was performed with peroxidase-labeled streptavidin biotin (LSA; DAKO, Glostrup, Denmark) using diaminobenzidine chromagen as substrate. All immunostaining was performed using the TechMate 500 (DAKO) automatic immunostaining device.

Epithelial cell selection/purification
Fresh samples of normal and tumor samples from 10 patients were disaggregated in a Medimachine instrument (DAKO Cytomation, Glostrup, Denmark). Cell viability was evaluated by trypan blue exclusion, all (>95%) of the cells in this fraction being viable. Epithelial cells were immunomagnetically purified from 2x106 total cells using superparamagnetic polystyrene beads coated with the Ber-EP4 antibody (specific for two glycopolypeptide membrane antigens expressed on most normal and neoplastic human epithelial tissues) (Dynabeds Epithelial Enrich; 1.5x106 beads in PBS/0.1% BSA/0.6% Na citrate; Dynal Biotech ASA, Oslo, Norway). mRNA was extracted from whole disaggregated samples (2x106 cells), from the epithelial cell-enriched fraction and from the supernatant containing non-epithelial cells. SNAIL and ZEB1 quantification was carried out in all of them. Owing to the high quality of mRNA isolated from viable cells in fresh tissue, in comparison with mRNA isolated from dead cells in frozen tissue, we detected SNAIL expression in normal and tumor tissues. Thus, expression in these samples was evaluated as a ratio (N/T). Cell nuclei were labeled with 4,2-diamidino-2-phenylindole dihydrochloride (DAPI) (SIGMA, Steinheim, Germany). Monoclonal antibodies against cytokeratin 8, 18 and 19, conjugated with FITC (DAKO Cytomation, Glostrup, Denmark), were used to label and visualize epithelial cells under the fluorescence microscope.

Statistical analysis
The following parameters were obtained from the medical records of the 114 patients: age, tumor size, lymph node metastases, pathological stage, histological grade, vascular invasion, polyps (defined by the presence of polyps in the surgery resection). Pathological stage was assessed using the tumor-node-metastases classification. Presence of lymph node metastases was evaluated by optical microscopy. No other immunohistochemical or molecular techniques were used.

The ratios (N/T) of gene expression were not normally distributed (Kolmogorov–Smirnov test, Lilliefors correction). Hence, we normalized the data distribution by using log10 for statistical analysis. We also used the geometric (rather than the arithmetic) average of the N/T to describe the expression gene data, for the same reason. When the distribution had been normalized, the variables analyzed, log(N/T), were contrasted by one-way ANOVA test.

The association between expression levels of different genes was analyzed by dividing the data into three groups with the same number of patients, by tertiles (33, 66%), and the result was contrasted by the {chi}2 test. The expression levels (N/T) defining the three groups for each gene were VDR, 1.20 (33%) and 3.30 (66%); CDH1, 0.54 (33%) and 2.24 (66%) and ZEB1, 0.67 (33%) and 5.12 (66%).

The multivariate analysis of the expression level of SNAIL and ZEB1 and the clinicopathological data was based on the logistic model. ZEB1 expression levels were divided in two groups using the median as the cut-off.

In all statistical tests, two-tailed P-values≤0.05 were considered statistically significant.

Statistical analysis was performed using SPSS version 11.0.


   ACKNOWLEDGEMENTS
 
We thank R. Rycroft for help with the English manuscript and M.E. Gómez for selection of tumor samples. This study was supported by Grants from Fundación Científica de la Asociación Española contra el Cáncer, from SAF2004-01015 and from Comunidad de Castilla la Mancha no. GC03012.

Conflict of Interest statement. The authors have declared no conflict of interest.


   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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