The Wnt antagonist sFRP1 is downregulated in premalignant large bowel adenomas

Abstract

Our previous studies have implicated the Wnt antagonist, sFRP1, as a tumour suppressor gene in advanced colorectal cancer. In this study, we set out to investigate the relationship between sFRP1 expression and large bowel adenomas, a precursor of colorectal cancer. The induction of beta-catenin/TCF mediated transcription is both a frequent early event in colorectal neoplasia, and a key downstream effect of wnt growth factor signalling. Lithium treatment of a small bowel mucosal cell line (FHs 74 int) induced sFRP1 within 8 h, indicating that this gene is positively regulated by beta-catenin, contrasting with the suppression of sFRP1 expression, we saw previously in advanced colorectal cancers. We therefore investigated a series of 12 adenomas and matched large bowel mucosa samples. Real-time RT-PCR analysis showed a reduction in sFRP1 expression in all 12 dysplastic lesions (median 485-fold, IQR 120- to 1500-fold), indicating factors other than beta-catenin influence sFRP1 levels. In a second series of 11 adenomas, we identified methylation of the sFRP1 promotor region in all 11 samples, and this was increased compared with the surrounding normal mucosa in seven cases. Immunohistochemical analysis using a polyclonal antibody supported these findings, with sFRP1 expression reduced in many of the adenoma samples examined. sFRP1 staining in normal mucosa adjacent to the dysplastic tissue was also reduced compared with the normal controls, suggesting that sFRP1 expression may be suppressed in a field of mucosa rather than in individual cells. This study identifies sFRP1 inactivation at the premalignant stage of colorectal cancer development, indicating that these pathways may be useful targets for chemoprevention strategies in this common solid tumour.

Figure 1

Real-time PCR quantitation of sFRP1 expression levels in a normal cell line following treatment with LiCl. (A) Real-time PCR quantitation of relative sFRP1 mRNA expression measured across a series of time points following lithium chloride treatment of the normal cell line FHS 74 Int according to the comparative C_T method. The initial step in the calculations is the normalisation of the sFRP1 gene to the KRT8 gene in order to normalise quantity and quality of the cDNA samples. The level of sFRP1 expression at each time point was then normalised to the level of sFRP1 expression before treatment. sFRP1 mRNA levels increased upon LiCl treatment peaking with an eight-fold increase at 8 h, which fell to six-fold at 16 h. (B) RT–PCR results for hNKD1 and β-actin at time 0 and 16 h.

Figure 2

Real-time PCR quantitation of relative sFRP1 mRNA expression in a series of 12 matched colorectal adenoma samples. Real-time RT–PCR was performed and the level of sFRP1 expression in each adenoma sample was compared to the results obtained for the adjacent matched normal tissue. The sFRP1 values are shown on a logarithmic scale. In all 12 adenoma samples, the sFRP1 mRNA levels were reduced substantially (>10 fold) with 83% of the samples showing a reduction of more than a 100-fold.

Figure 3

sFRP1 methylation status was analysed using the COBRA (COmbined Bisulfite Restriction Analysis) method as described previously (Caldwell et al, 2004). Methylated DNA remains capable of cleavage while unmethylated DNA is resistant. In total, 11 matched colorectal adenoma samples were analysed. The percentage of methylated DNA in each sample was measured by comparing the intensity of the ‘cut’ band against that of the ‘uncut’ band.

Figure 4

Immunohistochemistry and Western blot analysis using a Polyclonal sFRP1 antibody. (A) A Western blot containing lysate from HEK293 cells transiently transfected with full-length mouse sFRP1 and untransfected HEK293 cells was probed an sFRP1 antibody raised in rabbit. The antibody gave a single band at 37 kDa in the transfected HEK293 cell line (b) but no band in untransfected cell (a). (B) The same antibody was used to analyse sFRP1 protein expression and cellular distribution in a series of formalin fixed wax embedded sections taken from normal bowel mucosa and colorectal adenomas. All of the normal sections showed moderate to strong cytoplasmic, supranuclear staining (a and b) whereas 45% (10 out of 22) of the adenomas demonstrated either no staining (c) or mild staining (d).