Down-regulation of CDH1 is associated with expression of SNAI1 in colorectal adenomas.

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

List of primers and probes used used for qRT-PCR.

<p>List of primers and probes used used for qRT-PCR.</p

Correlation of Snail1 and E-cadherin protein expression in the immunochemistry.

<p>Y-axis: number of adenomas; white column = Snail1 negative; black column = Snail1 positive (p = 0.095).</p

E-cadherin expression in normal colonic mucosa and colorectal adenoma.

<p>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).</p

List of primary antibodies used for immunohistochemistry.

<p>List of primary antibodies used for immunohistochemistry.</p

Expression of CDH1 mRNA in correlation to SNAI1, TWIST1 and CDH2 mRNA occurrence.

<p>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. <b>A:</b> The amount of CDH1 mRNA was significantly lower in SNAI1 positive adenomas compared to SNAI1 negative ones (p = 0.004). <b>B:</b> TWIST1 positive adenomas had a reduced amount of CDH1 mRNA, but the difference to TWIST1 negative adenomas did not reach significance (p = 0.29). <b>C:</b> Co-expression of SNAI1 and TWIST1 showed a highly significant reduction in CDH1 mRNA (p = 0.003). <b>D:</b> Adenomas with expression of CDH2 mRNA did not show any significant difference in the amount of CDH1 mRNA compared to adenomas without CDH2 mRNA (p = 0.24).</p

CDH1 mRNA expression in colorectal adenoma, normal colonic mucosa and colorectal carcinoma.

<p>Y-axis: relative amount of CDH1 mRNA on a log-scale; x-axis: sample tissue. *p = 0.035. Bars indicate the median value.</p

Snail1 expression in normal colonic mucosa and colorectal adenoma.

<p>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.</p

Expression profile of colorectal adenomas in the qRT-PCR.

<p>CDH1: up- and downregulation compared to normal colonic mucosa.</p