Target gene activation of the Wnt-signalling pathway in the adamantinomatous subtype of craniopharyngiomas

Abstract

Objectives: Craniopharyngiomas (CPs) are rare, benign, epithelial tumours of the sellar region, histologically classified into adamantinomatous and papillary subtypes. The Wnt signalling pathway is involved in the pathogenesis of adamantinomatous CP, indicated by activating mutations within exon 3 of the β-catenin gene as well as nuclear accumulation of ß-catenin protein in 95% of respective tumours. However, several issues remain to be addressed, e.g. whether activating mutations in exon 3 induce nuclear β-catenin accumulation per se, or are other active and passive nuclear transport mechanisms also be involved (i.e. APC)? In addition, there is no proven evidence that nuclear ß-catenin activates target genes of the Wnt-signalling cascade in affected tumour cells. To answer these intriguing questions we performed a laser-microdissection based study comparing accumulating vs. non-accumulating tumour cells in adamantinomatous CPs. Mutation analysis as well as gene expression profiling with real-time PCR were conducted. Methods: Laser-microdissected cell clusters with or without β-catenin accumulation were subjected to mutational subsequent SSCP analysis of exon 3 of the β-catenin gene, in order to explore a potential genotypic heterogeneity (n=3). Furthermore, mutational analysis was extended towards exons 8 to 12 encoding armadillo repeats, which are crucial for passive nuclear transport of ß-catenin. Since active nuclear transport of ß-catenin is operated by APC, we have included an immunohistochemical analysis in 71 adamantinomatous and 16 papillary craniopharyngiomas using APC – specific antibodies. To adress the issue whether nuclear ß-catenin activates the Wnt-signaling cascade in adamantinomatous CPs, real-time RT-PCR analysis of conductin, a well recognised target gene and inhibitor of β-catenin, was examined in snap frozen tumour samples as well as in a laser – microdissected formalin fixed paraffin embedded tumour specimen. Results: Mutations in exon 3 of the β-catenin gene were identified in both, accumulating and non-accumulating cell populations. However, analysis of exons 8 to 12 did not reveal additional alterations in a series of 44 craniopharyngiomas. Whereas APC-mutations were not detectable in CP, our immunohistochemical analysis showed nuclear APC accumulation in 60 out of 71 (85%) adamantinomatous, and 6 out of 16 (38%) papillary CPs. A distinct overlap of nuclear β-catenin and APC expression was identified only in adamantinomatous CP. In addition, the β-catenin accumulating cell fraction revealed a 20 fold upregulation of conductin compared to non – accumulating adamantinomatous or papillary CPs. Conclusion: Our data confirm activating β-catenin mutations in exon 3 in adamantinomatous CPs. However, these mutations may not associate with nuclear β-catenin transfer per se, but rather point to the nuclear APC export machinery of β-catenin to be activated, but not genetically compromised. Immunohistochemical colocalisation of APC and ß-catenin within cell nuclei support this notion. Furthermore, nuclear β-catenin accumulation results in target gene activation and overexpression of inhibitors of the Wnt-signalling pathway i.e. conductin. The present results further highlight the Wnt-signaling pathway as molecular basis of the distinct and challenging clinical and morphological phenotype of adamantinomatous CPs.