Patterning and nuclear beta-catenin expression in the colonic adenoma-carcinoma sequence. Analogies with embryonic gastrulation

Abstract

Patterning is a process by which ordered arrangements of cells and tissue structure are attained. The term derived from developmental biology is also useful for the study of colonic carcinogenesis, in which the patterning of neoplastic tubules is necessary for properties of growth, invasion, and metastasis. Interestingly the nuclear expression and transcriptional activity of beta-catenin, a major oncoprotein in colonic carcinogenesis, is decisive for the first patterning of a tubule in embryogenesis, which creates the primitive gut and is called the gastrulation. Thus, basic patternings of embryogenesis and carcinogenesis might be linked. To test this hypothesis we compared morphological patterns and immunohistochemical beta-catenin stainings in colonic adenomas and adenocarcinomas with the gastrulation steps. Two analogies were found: 1) the patterning of invasion with reconstruction in adenocarcinomas corresponded to the epithelio-mesenchymal transition, ingression, and rearrangement of cells during the first phase of gastrulation; and 2) the patterning of tubular branching in adenomas and adenocarcinomas resembled the endodermal invagination during the second phase. The intratumorous distribution and intensity of nuclear beta-catenin expression was significantly correlated with the two patternings, similar to the findings in gastrulation. The results indicate microenvironmental regulations of nuclear beta-catenin expression and a return of neoplastic cells to embryonic transcriptional susceptibilities during colonic carcinogenesis.

Figure 1.

The patterning of tubular branching in adenomas is characterized by budding (a) or by ramification and extension (b) of epithelial cells from the tubules in H&E stains. Immunostaining by the monoclonal antibody for β-catenin demonstrates its nuclear expression in most of the budding epithelial cells, whereas nonbudding epithelium in the lower half of the picture contains only few positive cells (c). Original magnifications: ×40 (a and b), ×63 (c).

Figure 2.

Correlation between adenoma size and tubular branching. Box-plot charts (50% of values within the box; horizontal bar, median; vertical bar, range of values). Included are the P values of the correlations between the indicated branching scores. A P value <0.05 was considered significant.

Figure 3.

Comparison of the immunostaining for cytokeratin 18 showing different grades (scores) of tubular branching (a –c) with the immunostaining for β-catenin (monoclonal antibody) exhibiting different grades (scores) of positive nuclei (d–f) in three adenomas. The first adenoma contains very few branching tubules (score 1) (arrow) and few nuclei with β-catenin (score 1) (a and d). The second adenoma contains some branching tubules (score 2) and some β-catenin (score 2) expressing nuclei (b and e). The third adenoma contains many and repeatedly branching tubules (score 3) and most of the nuclei are β-catenin-positive (score 3) (c and f). Original magnifications: ×20 (a–c), ×40 (d–f).

Figure 4.

Spatial distribution and intensity of β-catenin expression, shown by immunostaining with the polyclonal antibody, correlate with the zonal organization and patterning in colonic adenocarcinoma. Zone of invasion with tubular reconstruction (a, short arrow, and higher magnification in c) with strongest nuclear β-catenin expression contains dissociated single tumor cells (c, small arrows), tumor cell plates and reconstructed tumor tubules. Zone of budding and tubular branching (a, long arrow, and higher magnification in b) exhibits many nuclei with weaker β-catenin expression at the budding sites. In continuity with branching tubules neoplastic epithelial cells with β-catenin at the adherens junctions and without nuclear expression occur (a, arrowhead, and higher magnification in b). Original magnifications: ×10 (a), ×20 (b and c).

Figure 5.

Epithelial-mesenchymal transition at the invasion front of a colonic adenocarcinoma. Immunostaining for fibronectin demonstrates neoplastic cells with cytoplasmatic fibronectin at the outer rim of a tumor cell complex (small arrows) and in dissociated, ingressing single tumor cells (arrows) within a strongly positive stroma (a). Immunostaining for E-cadherin detects strong membranous expression in tumor cell complexes, but loss of membranous positivity and weak cytoplasmic expression of a dissociated single cell in the stroma (arrowhead) (b). Original magnification, ×100.

Figure 6.

Double-immunoflourescence for E-cadherin (green) and fibronectin (red) in normal colonic mucosa (a) and at the invasion front of colonic adenocarcinoma (b). Yellow staining indicates a co-localization of membranous E-cadherin and membrane-bound fibronectin. Lack of cytoplasmic expression of fibronectin with clear separation of yellow-green membranous E-cadherin expressing cryptal epithelial cells and red fibronectin-positive lamina propria mucosae in normal mucosa (a). Green neoplastic cells with membranous and cytoplasmic E-cadherin expression (short arrow), red neoplastic cells with cytoplasmic fibronectin expression (long arrow) and yellow-orange cells with mixed fibronectin and E-cadherin expression (arrowhead) at the invasion front. Original magnification, ×40.

Figure 7.

Schematic demonstration of the analogies of patterning and nuclear β-catenin expression in the sea urchin gastrulation and colonic neoplasms. The first phase of gastrulation with epithelio-mesenchymal transition and mesenchymal ingression at the vegetal plate of the blastocyste corresponds with the patterning of invasion with tubular reconstruction in colonic adenocarcinomas. Strong nuclear β-catenin expression (dark red) is found. The second phase of gastrulation with endodermal invagination comprising a budding and tubular extension is similar to the patterning of tubular branching. Here a weaker nuclear β-catenin expression (light red) is seen.

Figure 8.

A hypothetical model for the morphogenesis of colonic adenocarcinoma derived from the analogy of the patterning with the gastrulation in developmental biology. Nuclear β-catenin might initiate a signal and transcriptional susceptibility of tumor cells for differentiation programs either toward an epithelial phenotype for position formation and tubular patterning or toward a mesenchymal phenotype with the properties for invasion with migration and metastasis. The direction of transcriptional activity and differentiation could be determined by contrarotating signaling gradients that could regulate the equilibrium of epithelio-mesenchymal transitions and thus the tumor behavior.