The authors have declared that no competing interests exist.
Conceived and designed the experiments: NHS FK GEWF WTK SEB. Performed the experiments: ZT CV MS GEWF FK SAT. Analyzed the data: NHS SAT JS WTK CV FK GEWF SEB MS GEWF. Contributed reagents/materials/analysis tools: NHS FK JS SAT SEB MS WTK. Wrote the paper: NHS FK GEWK SEB JS. Shared first authorship: FK GEWF.
Down-regulation of E-cadherin (CDH1) and epithelial-mesenchymal transition (EMT) are considered critical events for invasion and metastasis of colorectal carcinoma. Here we tested whether the important regulators of E-cadherin expression SNAI1 and TWIST1 are already detectable in human colorectal adenomas.
RNA was extracted from a set of randomly selected formalin-fixed and paraffin-embedded (FFPE) colorectal adenomas (n = 41) and normal colon mucosa (n = 10). Subsequently mRNA expression of CDH1, CDH2, SNAI1 and TWIST1 was analysed by quantitative RT-PCR analysis. CDH1 as well as SNAI1 protein expression were assessed by immunohistochemistry (IHC).
SNAI1 mRNA was expressed in 78% (n = 32/41), TWIST1 mRNA in 41% (n = 17/41) and CDH2 mRNA in 41% (n = 17/41) of the colorectal adenoma tissue, while normal colon mucosa was negative for these transcription factors. We found a significant correlation between reduced CDH1 and the presence of SNAI1 mRNA expression and for combined SNAI1 and TWIST1 mRNA expression, respectively. A correlation between CDH2 mRNA expression and reduced CDH1 expression was not observed. We confirmed the relationship between SNAI1 expression and reduced E-cadherin expression on the protein level via IHC.
Our data show that SNAI1 and Twist1 are already expressed in benign precursor lesions of colorectal cancer and that SNAI1 expression was significantly correlated with lower expression of CDH1. Whether these findings reflect true EMT and/or are a sign of a more aggressive biology need to be investigated in further studies.
Epithelial-mesenchymal transition (EMT) denotes a process in which cells change their phenotype between epithelial and mesenchymal states. This phenotypic change involves complex molecular and cellular programs by which epithelial cells can dispose of their differentiated characteristics, including cell-cell adhesion, planar and apical-basal polarity, lack of motility and gain instead mesenchymal features such as motility, invasiveness and increased apoptotic resistance
Ostensibly, the ‘uncontrolled’ reactivation of such EMT programs occurs frequently in cancer cells
In order to investigate, whether the EMT “master regulators” SNAI1 and TWIST1 and the mesenchymal marker CDH2 are already expressed in colorectal adenomas, we assessed their expression in formalin fixed and paraffin embedded (FFPE) tissues and used previously published primers and probes for a quantitative RT-PCR assay (qPCR) that were shown to work well in FFPE material
Colorectal adenoma specimens obtained in the period 2002 to 2007 were retrieved from the files of the Department of Pathology (University Hospital Düsseldorf). All patients suffering from known hereditary colorectal cancer syndromes were excluded. A total of 41 benign colorectal adenoma specimens of 35 patients were randomly selected. In addition, normal colonic mucosa (n = 10) and colorectal cancer tissue (n = 10) from the same period were selected. This study was approved by the Ethics Committee of the Medical Faculty of the Heinrich-Heine University Düsseldorf, they waived the need for written informed consent for using the patients' material, as it was analysed anonymously. This is also in accordance to the recommendation of the German Central Ethics Committee from 2003.
Our series consisted of 63% (n = 22) male and 37% (n = 13) female patients; the average age at the moment of resection was 68 years. All adenomas were graded and classified according to histologic type and degree of intraepithelial neoplasia by experienced pathologists.
Adenoma | Case | Sex | Age | Size | Histology | Dysplasia |
1 | 1 | M | 78 | 0,7 | tubular | low grade |
2 | 2 | M | 51 | 0,7 | tubular | low grade |
3 | 3 | F | 76 | 1,5 | tubulovillous | low grade |
4 | 4 | M | 76 | 0,2 | tubular | low grade |
5 | 5 | F | 86 | 0,2 | tubular | low grade |
6 | 6 | M | 74 | 2,0 | tubulovillous | low grade |
7 | 7 | F | 41 | 0,2 | tubular | low grade |
8 | 8 | M | 62 | 1,4 | tubulovillous | low grade |
9 | 9 | M | 73 | 3,0 | tubulovillous | low grade |
10 | 10 | M | 72 | 0,5 | tubulovillous | low grade |
11 | 10 | M | 72 | 0,5 | tubular | low grade |
12 | 11 | F | 85 | 2,0 | tubulovillous | low grade |
13 | 12 | M | 68 | 0,5 | tubular | low grade |
14 | 13 | M | 60 | 3,0 | tubular | low grade |
15 | 14 | M | 79 | 0,5 | tubular | low grade |
16 | 14 | M | 79 | 1,0 | tubular | low grade |
17 | 14 | M | 79 | 1,5 | tubular | low grade |
18 | 14 | M | 79 | 5,0 | tubular | high grade |
19 | 15 | F | 71 | 4,2 | tubulovillous | low grade |
20 | 16 | M | 54 | 0,2 | tubular | low grade |
21 | 17 | M | 68 | 0,5 | tubular | low grade |
22 | 18 | F | 58 | 0,5 | tubular | low grade |
23 | 19 | F | 64 | 0,5 | tubular | low grade |
24 | 20 | M | 60 | 0,9 | tubulovillous | low grade |
25 | 21 | M | 51 | 1,5 | tubulovillous | low grade |
26 | 22 | M | 68 | 0,5 | tubular | low grade |
27 | 23 | M | 63 | 1,5 | tubulovillous | low grade |
28 | 23 | M | 63 | 3,0 | tubulovillous | high grade |
29 | 24 | F | 65 | 0,7 | tubular | low grade |
30 | 24 | F | 65 | 1,1 | tubular | high grade |
31 | 25 | F | 83 | 1,0 | tubular | low grade |
32 | 26 | M | 78 | 1,5 | tubulovillous | low grade |
33 | 27 | M | 97 | 4,5 | tubular | high grade |
34 | 28 | M | 63 | 8,0 | tubulovillous | high grade |
35 | 29 | F | 72 | 1,2 | tubulovillous | high grade |
36 | 30 | M | 60 | 1,0 | tubular | high grade |
37 | 31 | F | 75 | 2,3 | tubular | high grade |
38 | 32 | F | 75 | 0,5 | tubular | high grade |
39 | 33 | M | 75 | 1,7 | tubular | high grade |
40 | 34 | M | 54 | 3,0 | tubular | high grade |
41 | 35 | F | 46 | 0,6 | tubular | high grade |
Size: greatest possible diameter in centimetres.
Total RNA was extracted from 4 µm serial sections of formalin-fixed paraffin-embedded (FFPE) specimens. Paraffin was removed by extracting two times with xylene for 5 minutes followed by rehydration through a graded ethanol series (100, 95, 70% ethanol). After the final 70% ethanol wash and subsequent rinsing in phosphate buffered saline (PBS, pH 7.4), the specimens were immersed in 3% glycerol for 30 seconds. Microdissection was carried out using sterile equipment and samples were transferred in a sterile 1.5-ml tube containing 1000 µl TRIzol reagent (Invitrogen, UK). Lysis was carried out at room temperature for at least 10 minutes or until the tissue was completely solubilized. The RNA was purified by chloroform extraction, followed by precipitation with an equal volume of isopropanol at room temperature. The RNA pellet was washed once with 75% ethanol, air-dried, and re-suspended in 30 µl of RNase-free water. The total RNA and a human reference RNA (1 ng/µl) (Clon tech, Canada) were reverse-transcribed in a total volume of 20 µL consisting of: 2 µl random hexamere primer, 20 U Protector RNase-Inhibitor, 2 µl dNTPs (10 mmol/L), 0.5 µl reverse transcriptase and 11 µl total RNA (containing a maximum of 2 µg RNA) in 5x RT-buffer (all: Roche Diagnostics, Germany). The reaction mixture was incubated at 25°C for 10 min followed by 30 min at 55°C. The enzyme inactivation step was carried out for 5 min at 85°C. The cDNA was stored at −20°C until use.
Quantitative RT-PCR analyses for SNAI1, TWIST1, CDH1, CDH2 and GAPDH were performed using the Dyad Disciple Chromo 4 instrument and software (BioRad, Germany). Intron-spanning primers and probes for the TaqMan system were selected from literature
Transcript | Sequence | |
GAPDH | Reverse | 5′-GCC ATC ACG CCA CAG TTT C-3′ |
Forward | 5′-CGT GGA AGG ATC CAT GAC CA-3′ | |
Probe |
|
|
CDH1 | Reverse |
|
Forward |
|
|
Probe |
|
|
SNAI1 | Reverse | 5′-GTG GGA TGG CTG CCA GC-3′ |
Forward |
|
|
Probe | 5′-TCC AGC AGC CCT ACA CCA GGC C-3′ | |
TWIST1 | Reverse | 5′-TGT CCA TTT TCT CCT TCT CTG GA-3′ |
Forward |
|
|
Probe | 5′-TCA GCA GGG CCG GAG ACC TAG ATG T-3′ | |
CDH2 | Reverse | 5′ - TC |
Forward | 5′ - GA |
|
Probe | 5′ - ACC |
Relative expression levels of target sequences were determined by the comparative Ct method (2−ΔΔCt) using GAPDH as housekeeping gene and a human reference RNA as external calibrator. We normalized the resultant Ct-values to the housekeeping gene, thus creating ΔCt-values (ΔCt = Ct(target)−Ct(housekeeping)). In a next step, we calculated the ΔΔCt-values, using the equation ΔΔCt = ΔCt(target)−ΔCt(calibrator). ΔCt(target) is the Ct-value of any target gene normalized to the endogenous housekeeping gene and ΔCt(calibrator) is the Ct-value of the same gene in the human reference RNA also normalized to the endogenous housekeeping gene. For all probes and primer pairs we determined the efficiency by the standard curve method. Since the efficiencies of our probes and primer pairs were approximately equal, we could use the comparative equation 2−ΔΔCt to calculate the relative amount of target gene
We used tissue from the same blocks as for the qRT-PCR, except for one case in which no further tissue was available. For immunostaining, 4 µm serial sections were stained as described in
Antigen | Antibody | Concentration | Animal source |
E-Cadherin | NCH-38, DAKO | 2 µg/ml | Mouse |
Snail1 | Ab17732, AbCam | 1 µg/ml | Rabbit |
Isotype control | MOPC-21, Sigma | 2 µg/ml | Mouse |
Isotype control | X0903, DAKO | 1 µg/ml | Rabbit |
Two independent observers examined the sections. Normal colonic mucosa adjacent to the adenomas was used as internal control. For E-cadherin, we used a scoring system that included an evaluation of both the staining intensity and the percentage of stained cells similar to Blechschmidt et al.
Statistical analysis was performed with the SPSS software (SPSS Standard version 17.0.0, SPSS Inc., Chicago, IL). Significance of differences between groups with a nonparametric data distribution was analyzed with the Mann–Whitney U test for two independent groups. The threshold for statistical significance was chosen at p<0.05.
We performed a quantitative RT-PCR assay to analyze the mRNA expression of SNAI1 (Snail1), TWIST1 (Twist1), CDH1 (E-cadherin) and CDH2 (N-cadherin) in FFPE tissue samples of colorectal adenomas (n = 41), colorectal cancer (n = 10) and normal colon mucosa (n = 10). In normal colonic mucosa, SNAI1, TWIST1 and CDH2 mRNA could not be detected at all. We could detect SNAI1 mRNA expression in 32 (78%) and TWIST1 mRNA expression in 17 (42%) of 41 colorectal adenomas. 13 out of 41 (32%) colorectal adenomas expressed both SNAI1 and TWIST1 mRNA at the same time. In 17 of the 41 (42%) colorectal adenomas, we detected CDH2 mRNA
CDH1: up- and downregulation compared to normal colonic mucosa.
CDH1 mRNA expression was detected in 39 of 41 (95%) colorectal adenoma samples, in all 10 (100%) colorectal cancer samples and in all 10 (100%) normal colonic mucosa samples. CDH1 expression was significantly reduced in colorectal adenomas compared to normal colonic mucosa (p = 0.035, Mann-Whitney-U test). As expected, expression of CDH1 mRNA was clearly decreased in colorectal cancer compared to the expression in normal colonic mucosa. Due to the small number of samples, this correlation did not reach significance (p = 0.199, Mann-Whitney-U test). The amount of CDH1 mRNA did not differ significantly between colorectal adenoma and colorectal cancer
Y-axis: relative amount of CDH1 mRNA on a log-scale; x-axis: sample tissue. *p = 0.035. Bars indicate the median value.
Next, we tested whether the expression of SNAI1 and TWIST1 mRNA correlated with the expression of CDH1 mRNA. When comparing the amount of CDH1 mRNA in colorectal adenomas with or without SNAI1 expression, we found a significant difference. In the 32 colorectal adenomas, which were positive for SNAI1 mRNA, the amount of CDH1 mRNA was significantly lower (p = 0.004, Mann-Whitney-U test), compared to the amount in colorectal adenomas without SNAI1 mRNA expression
See Methods for details on qRT-PCR and quantification. Y-axis: relative amount of CDH1 mRNA on a metric scale; X-axis: adenomas positive or negative for target transcript. Boxed regions enclose 25th to 75th percentiles, with the horizontal line indicating the median. Whiskers include 5th to 95th percentiles.
Even though it was often proposed that N- and E-cadherin have contrary functions in cancer progression
CDH1: up- and downregulation compared to normal colonic mucosa.
We did not detect any correlation between the genes of interest and the histologic type (tubular or tubulovillous adenoma), the grading (low or high grade) or the size (<1 cm or ≥1 cm) of the adenomas. Also there was no correlation to the age at diagnosis (<68 years or ≥68 years) or the gender of the patients.
To correlate our qRT-PCR results on the protein level, we performed an immunohistochemical analysis of Snail1 and E-cadherin protein in 40 of the colorectal adenomas. For Snail1, only detectable nuclear staining was considered positive
Expression of Snail1 was determined as indicated in Methods using Ab17732 antibody as positive and X0903 antibody as negative control. Panels A and B show corresponding areas of a colorectal adenoma. Panel A corresponds to Snail1 staining (arrows = Snail1 positive cells), while panel B shows the negative control. A1: adenomatous tissue negative for Snail1 staining. A2: colorectal adenoma tissue positive for nuclear Snail1 staining Panels B1 and B2: no positive reaction in negative control.
Expression of E-cadherin was determined as indicated in Methods using NCH-38 antibody and MOPC-21 as isotype control. Panels A and B show normal colonic mucosa and colorectal adenoma tissue (respectively). Note the difference in E-cadherin expression. The inlays in panels A and B correspond to the negative control of the same sample. Panel C shows an overview of a colorectal adenoma with adjacent normal colonic mucosa. C1 and C2 correspond to the indicated areas in panel C and show normal colonic mucosa with normal E-cadherin staining (C1) and colorectal adenoma with reduced E-cadherin staining (C2).
The Snail1 immunohistochemistry correlated significantly with the level of CDH1 mRNA (p = 0.02, Mann-Whitney-U test). Adenomas with positive Snail1 nuclear immunostaining had a lower level of CDH1 mRNA and with absent nuclear Snail1 staining showed higher levels of CDH1 mRNA
The colorectal adenomas with preserved E-cadherin staining showed a significantly higher amount of CDH1 mRNA in the qRT-PCR, compared with colorectal adenomas with reduced E-cadherin immunoreactivity (p = 0.003, Mann-Whitney-U test)
Y-axis: relative amount of target mRNA in the qRT-PCR on a log.scale; X-axis: positive or negative immunostaining for target protein. Bars indicate median value.
On the transcriptional level, we observed a significant correlation between SNAI1/Snail1 expression and CDH1/E-cadherin loss in colorectal adenomas
Y-axis: number of adenomas; white column = Snail1 negative; black column = Snail1 positive (p = 0.095).
It has been clearly shown in a variety of model systems that cancer cells use EMT to down-regulate their cell-cell contacts and to become motile and invasive
For our qPCR-assay we used primers and probes published by Rosivatz et al.
According to our data, SNAI1, but not TWIST1, seems to contribute significantly to the down-regulation of E-cadherin in benign colorectal adenomas, in which decreased E-cadherin levels have already been described
A potentially contradictory result of our study was the noted co-expression of CDH1/E-cadherin and CDH2. This observation is however consistent with findings by Rosivatz et al
In conclusion, our hypothesis generating study revealed SNAI1 expression as well as combined SNAI1/TWIST expression to be associated with decreased expression of CDH1 in colorectal adenomas. Whether the expression of the EMT transcription factors has an influence on the malignant potential of the colorectal adenomas was not addressed in our study. However, it is of interest that a recent transcriptome profiling study comprising over 320 CRC revealed an EMT-signature as the dominant pattern of intrinsic gene expression. This EMT-signature was tightly correlated with shortened relapse-free survival. Major components of the signature were up-regulated TWIST and down-regulated CDH1
We thank Imke Hoffmann, Swetlana Seidschner and Sarah Schumacher for suggestions and technical assistance.