Target gene activation of the Wnt signaling pathway in nuclear beta-catenin accumulating cells of adamantinomatous craniopharyngiomas

Abstract

Activating beta-catenin (CTNNB1) mutations can be identified in the majority of adamantinomatous craniopharyngiomas (adaCP), suggesting an aberrant Wnt signaling pathway in this histopathologically peculiar tumor entity. However, there is no proven evidence that nuclear translocation of beta-catenin is associated with CTNNB1 mutations and target gene activation. We performed a laser-microdissection-based study comparing beta-catenin accumulating vs. non-accumulating tumor cells. Mutational analysis and gene expression profiling using real-time polymerase chain reaction were conducted in adamantinomatous and papillary tumor specimens. Target gene activation, that is, over-expression of Axin2 could be detected in adaCP, especially in tumor cells with nuclear beta-catenin accumulation. In addition, increased expression of BMP4 was identified in the accumulating cell population, which supports the hypothesis of an oral ectodermal origin. Interestingly, accumulating and non-accumulating tumor cell populations carried CTNNB1 mutations within exon 3. We extended the analysis, therefore, towards genetic regions encoding for membrane linkage and active/passive nuclear transport mechanisms (exon 4 and exon 8-13), but could not detect any alteration. This is the first report demonstrating an association between nuclear beta-catenin accumulation and target gene activation in adaCP. The results confirm the Wnt signaling pathway as molecular basis of the distinct and challenging clinical and morphological phenotype of adaCP.

Figure 1

Molecular‐genetic analysis of laser‐microdissected tumor cells. For mutational analysis, we separated nuclear β‐catenin accumulating (+) vs. non‐accumulating (−) tumor cell fractions using laser microdissection. The arrows indicate accumulating cell clusters before and after dissection (A). Direct sequencing of exon 3 of CTNNB1 revealed a missense mutation at codon 37 encoding for a serine residue and phosphorylation site of GSK‐3β kinase in both fractions [accumulating (+) and non‐accumulating (−)] (B).

Figure 2

Elevated levels of Axin2/conductin and BMP4 in total tumor cDNA of adaCP vs. papCP as well as in accumulating vs. non‐accumulating tumor cell mRNA. A. Relative quantification TaqMan analysis of Axin2/Conductin and BMP4 cDNA obtained from snap frozen and histologically confirmed papCP (n = 5) and adaCP (n = 5), harboring a sufficient amount of β‐catenin accumulating tumor cell clusters. β‐2‐microglobulin served as endogenous control and papCP were used as calibrator. All analyses were performed in quadruplicates of each sample. B. mRNA expression of β‐catenin target genes Axin2/Conductin as well as BMP4 is increased in laser‐microdissected tumor cell clusters with nuclear β‐catenin accumulation (+) in relation to non‐accumulating cells (−) (TaqMan analysis). The experiments were conducted three times.

Figure 3

Immunohistochemical co‐localization of Axin2/BMP4 and β‐catenin. Using serial sections, whorl‐like tumor cell clusters showing nuclear β‐catenin accumulation (A,C), displayed elevated levels of Axin2 (B) and BMP4 (D). Magnification is 200× for A,B and 400× for C,D.