Novel insights into Notum and glypicans regulation in colorectal cancer

Abstract

The connection between colorectal cancer (CRC) and Wnt signaling pathway activation is well known, but full elucidation of the underlying regulation of the Wnt/β-catenin pathway and its biological functions in CRC pathogenesis is still needed. Here, the azoxymethane/dextran sulfate sodium salt (AOM/DSS) murine model has been used as an experimental platform able to mimic human sporadic CRC development with predictable timing. We performed genome-wide expression profiling of AOM/DSS-induced tumors and normal colon mucosa to identify potential novel CRC biomarkers. Remarkably, the enhanced expression of Notum, a conserved feedback antagonist of Wnt, was observed in tumors along with alterations in Glypican-1 and Glypican-3 levels. These findings were confirmed in a set of human CRC samples. Here, we provide the first demonstration of significant changes in Notum and glypicans gene expression during CRC development and present evidence to suggest them as potential new biomarkers of CRC pathogenesis.

Keywords: Notum; WNT-pathway; colorectal carcinogenesis; glypicans; predictable animal models.

Figure 1. Experimental procedure and macroscopic and histological observation of the AOM/DSS murine model

A. Schematic experimental procedure for groups treated with AOM-alone and/or DSS. Control group (untreated littermate controls) not represented. B. Macroscopic observation of the distal regions of colons from control, AOM-, DSS- and AOM/DSS-treated mice at the end of the 20^th week (only 3 of 6 animals per group are shown). Evident macroscopic lesions detectable only in AOM/DSS-treated colons. C. Hematoxylin/eosin staining of tumors and normal colons. Colon mucosae of AOM-only and DSS-only treated mice show the same histological characteristics of the control group. Adenocarcinomas with a high degree of dysplasia are detectable in AOM/DSS-treated mice. 20x original magnification. Scale bar, 50 μm.

Figure 2. Hierarchical clustering of gene expression data

Hierarchical clustering was generated by R hclust function, using Euclidean distance and average linkage as metrics.

Figure 3. Multi-Gene qPCR validation of differential gene expression

Gene expression levels were measured in the four different conditions: Untreated (white circle), DSS (grey square), AOM (blue square) and AOM/DSS (red circle). The results are expressed as Delta C_T values between the C_T value of the gene of interest and the C_T value of β2-microglobulin. Each dot represents the evaluation of the gene levels in a single mouse. Statistically significant differences were calculated using Student's t-test: ***p < 0.0005; **p < 0.0078.

Figure 4. Enriched canonical pathways of the differentially expressed genes as determined by Ingenuity Pathway Analysis (IPA)

The significance of canonical pathways was determined by IPA's default threshold [−log(p-value)]> 3 for adenocarcinoma and [−log (p-value)] > 1.3 for colon mucosa of AOM-only and DSS-only treated mice. P-value calculated by Fisher's exact test. The associated gene number above each column represents the number of differentially expressed genes that were involved in the respective canonical pathways. The percentage of genes that were up- or down-regulated is represented in red or green, respectively. In the lower figure, the Wnt/β-catenin pathway has been added and shows the IPA overlay of analysis in adenocarcinoma (up-regulated genes in red, down-regulated genes in green). Direct or indirect interactions are shown by complete or dashed lines, respectively.

Figure 5. Representative results of immunostaining for β-catenin, Notum, Glypican-1, Glypican-3 in FFPE mouse tissue sections

Membrane-bound β-catenin, Notum and Glypican-1 were observed in normal colon mucosa, whereas dysplastic ACF (Aberrant Crypt Foci), microadenomas, and adenocarcinomas exhibited more intense (++) staining (nuclear or cytoplasmic). Glypican-3 staining was less intense (+) than Glypican-1 staining (++), and the adenocarcinoma showed a negative signal (−) with respect to nuclear or cytoplasmic compartments along with more intense (++) extracellular staining. Incubation with a primary antibody was omitted in the negative control. Sections were counterstained with hematoxylin: 20x and 40x original magnification. Scale bar, 50 μm.

Figure 6. A. qPCR analysis of NOTUM, GPC1 and GPC3 differential gene expression in human samples

Gene expression levels were measured in human colorectal adenocarcinomas with respect to normal colon mucosae, and results are expressed as fold changes, considering the C_T value of the gene of interest and the C_T value of β-actin. Data are represented as mean +/− SD). Statistically significant differences were calculated using Student's t-test: ***p < 0.0001; **p < 0.001. B. Representative results of immunostaining for Notum, Glypican-1 and Glypican-3 in human tissue sections. Strong staining (++) in CRC cases for Notum and Glypican-1; weak staining (−) in CRC cases for glypican-3. Incubation with a primary antibody was omitted in the negative control. Sections were counterstained with hematoxylin: 20x and 40x original magnification. Scale bar, 50 μm.