Expression of Wnt ligands and Frizzled receptors in colonic mucosa and in colon carcinoma

Abstract

Aims: Signalling through the Wnt pathway is integrally associated with colon carcinogenesis. Although activating mutations in the genes for adenomatous polyposis coli (APC) and beta-catenin are clearly associated with colon cancer, less is understood about the role of the upstream secreted ligands (Wnts) and their receptors (frizzled, Fz) in this process. In other systems, increased Wnt signalling has been shown to alter the expression of components of this pathway. This study was designed to test the hypothesis that colon cancer is characterised by aberrant expression of specific Wnt genes and Fz receptors.

Methods: The expression of Wnt genes was assessed by in situ, antisense RNA hybridisation in paraffin wax embedded samples of normal and malignant human colon tissues with probes specific for the individual Wnt genes. The expression of Fz1 and Fz2 was determined by immunoperoxidase based antibody staining on human tissues.

Results: Changes in the expression of some ligands and receptors were seen in colon cancer. For example, Wnt2 mRNA was detected in colon cancer but was undetectable in normal colonic mucosa. Differential expression of Wnt5a in normal mucosa was also noted, with increased expression at the base of the crypts compared with the luminal villi and slightly increased expression in colon cancer. Wnt7a exhibited minimal expression in both normal and malignant colon tissues, whereas other Wnt ligands including Wnts 1, 4, 5b, 6, 7b, and 10b were expressed equally and strongly in both normal and malignant colon tissues. In defining cellular responses and phenotype, the type and distribution of Fz receptors may be as important as the pattern of Wnt ligand expression. No expression of Fz receptor 1 and 2 was seen in normal colonic mucosa and in well differentiated tumours. However, poorly differentiated tumours exhibited a high degree of Fz receptor expression, especially at the margin of cellular invasion.

Conclusions: These data indicate that the expression of members of the Wnt signal transduction pathway, distinct from APC and beta-catenin, is integrally associated with the process of colon carcinogenesis. Wnt2, and possibly Wnt5a, may be involved in the progression from normal mucosa to cancer and the expression of Fz1/2 receptors may be involved in processes associated with tumour invasion. Altered expression of these Wnts and Fz receptors may prove useful as prognostic or diagnostic markers for patients with colon cancer.

Figure 1

In situ, anti-sense RNA hybridisation of Wnt5b in HT29 colon cancer cell lines. In situ hybridisation of paraffin wax embedded cells. Dark colouration indicates positive hybridisation. Inserts represent control hybridisation with sense orientation Wnt5b probe in HT29 cells. (A) Original magnification, ×100; (B) original magnification, ×400.

Figure 2

Expression, as measured by in situ antisense RNA hybridisation, of Wnt 2 (top row) and bone morphogenetic protein 6 (BMP6; bottom row) in normal colon (left two columns, original magnifications, ×40 and ×100) and colon cancer (right column, original magnification, ×100) from the same individual. The dark purple colouration indicates positive hybridisation. The pink staining is the result of the FAST red dye used to reveal tissue architecture. Hybridisation for a specific probe on normal and cancer tissues was performed concomitantly, under identical conditions, and with freshly prepared probe.

Figure 3

Expression, as assessed by antibody based immunoperoxidase methodology, of Fz1/2 receptors in (A1–3) normal colonic mucosa, (B1–3) well differentiated colon carcinoma, and (C1–3) poorly differentiated colon carcinoma. Haematoxylin and eosin staining is depicted in A1, B1, and C1. Experimental conditions are represented in panels A2, B2, and C2 (original magnification, ×40) and A3, B3, and C3 (original magnification, ×100). The brown granular staining in panels C2 and C3 indicates the presence of Fz1/2 receptors.

Figure 4

Diagram representing the expression of various Wnt ligands, BMP2a, and BMP6 in the normal colon and (B) the comparative expression of these molecules in normal and malignant colon.