Identification of novel pathways involved in the pathogenesis of human adamantinomatous craniopharyngioma

Abstract

Activating mutations in the gene encoding β-catenin have been identified in the paediatric form of human craniopharyngioma (adamantinomatous craniopharyngioma, ACP), a histologically benign but aggressive pituitary tumour accounting for up to 10% of paediatric intracranial tumours. Recently, we generated an ACP mouse model and revealed that, as in human ACP, nucleocytoplasmic accumulation of β-catenin (β-cat(nc)) and over-activation of the Wnt/β-catenin pathway occurs only in a very small proportion of cells, which form clusters. Here, combining mouse genetics, fluorescence labelling and flow-sorting techniques, we have isolated these cells from tumorigenic mouse pituitaries and shown that the β-cat(nc) cells are enriched for colony-forming cells when cultured in stem cell-promoting media, and have longer telomeres, indicating shared properties with normal pituitary progenitors/stem cells (PSCs). Global gene profiling analysis has revealed that these β-cat(nc) cells express high levels of secreted mitogenic signals, such as members of the SHH, BMP and FGF family, in addition to several chemokines and their receptors, suggesting an important autocrine/paracrine role of these cells in the pathogenesis of ACP and a reciprocal communication with their environment. Finally, we highlight the clinical relevance of these findings by showing that these pathways are also up-regulated in the β-cat(nc) cell clusters identified in human ACP. As well as providing further support to the concept that pituitary stem cells may play an important role in the oncogenesis of human ACP, our data reveal novel disease biomarkers and potential pharmacological targets for the treatment of these devastating childhood tumours.

Fig. 1

Purification of β-catenin-accumulating cells from Hesx1 ^Cre/+;Ctnnb1 ^lox(ex3)/+;BAT-gal pituitaries. a X-gal staining of a Hesx1 ^Cre/+;Ctnnb1 ^lox(ex3)/+;BAT-gal mutant pituitary showing the activation of the BAT-gal reporter and expression of β-galactosidase in clusters within the anterior lobe. The posterior lobe is highly stained and was surgically removed prior to the purification procedure. b Double immunofluorescence staining against β-catenin (green) and SOX2 (red) in wild-type and Hesx1 ^Cre/+;Ctnnb1 ^lox(ex3)/+ (mouse ACP) pituitaries. Note the nucleocytoplasmic accumulation of β-catenin in very few cells that form clusters in the Hesx1 ^Cre/+;Ctnnb1 ^lox(ex3)/+ pituitary (arrows), whilst the majority of the cells show normal cytoplasmic staining as in the control pituitary. SOX2 is expressed in cells of the marginal zone around the lumen in the control but in Hesx1 ^Cre/+;Ctnnb1 ^lox(ex3)/+ pituitaries, some cells within the clusters also express SOX2 (arrows). Note a cluster that is negative for SOX2 expression (arrowhead). c Scheme of the strategy to purify the β-catenin-accumulating cell clusters. Triple heterozygous pituitaries (only anterior and intermediate lobes) are dissociated into single cell suspension, treated with CMFDG (a fluorogenic substrate for β-galactosidase) and subjected to flow-sorting. The two fractions are used for qRT-PCR analysis, gene profiling and stem cell culture. d qRT-PCR comparing BAT-gal^+ve (clusters) versus BAT-gal^−ve (non-clusters) cell fractions confirming the efficiency of the purification. Fold-changes in expression indicated on the y-axis, where >0 means higher expression in BAT-gal^+ve and <0 higher in BAT-gal^−ve. Cluster cells fit an undifferentiated profile (high Sox2 and low Pit1, Pomc1 and Gh) and show activation of the Wnt/β-catenin pathway (high lacZ, Axin2, Lef1 and CyclinD1). e, f The BAT-gal^+ve fraction contains 8.48 times more cells with clonogenic potential (progenitors/stem cells, PSCs) (e) able to form single cell-derived colonies when cultured in stem cell-promoting media (f). pl posterior lobe, il intermediate lobe, al anterior lobe, mz marginal zone. Scale bars b 50 μm, f 100 μm

Fig. 2

Pituitary progenitors/stem cells (PSCs) are contained in the SOX2-expressing population. a Scheme of cell purification strategy: Sox2 ^eGFP/+ postnatal pituitaries (P14) are dissociated into single cell suspensions and flow sorted to separate the eGFP^+ve and eGFP^−ve fractions. b Scatter plots showing the isolation of the two fractions. The gates used are indicated on the plot. c Clonal culture of cell preparations from unpurified Sox2 ^eGFP/+ pituitaries in stem cell-promoting media gives rise to eGFP^+ve colonies demonstrating the activation of the SOX2 promoter in PSCs. d Photograph of a tissue culture plate containing fixed and hematoxylin-stained colonies demonstrate the presence of colonies only in the flow sorted purified eGFP^+ve fraction. e Approximately 2.4% of the eGFP (Sox2)-expressing cells are able to form colonies. Scale bar 50 μm

Fig. 3

Analysis of telomere length in β-catenin accumulating clusters and surrounding cells in mouse and human ACP. (a, b) Telomere PNA FISH (Cy3 conjugate) and β-catenin immunofluorescence on mouse pre-tumoral lesions at 18.5 dpc (a) and human ACP samples (b) allows visualisation of telomeres specifically in β-catenin accumulating cluster cells or non-cluster surrounding cells. c, d Quantification of telomere length as sum spot intensity reveals significantly longer telomeres in β-catenin accumulating cluster cells in the mouse (c) whereas these are significantly shorter in the advanced human ACP sample (d). The pictures shown (a, b) are representative examples corresponding to single optical confocal sections of 0.001 μm thickness. Indicated statistics prepared using a Students t-test, n = 50 clusters for mouse, n = 25 clusters for human. Scale bar 50 μm

Fig. 4

SHH signalling is active in mouse and human ACP. a Double immunofluorescence using antibodies against β-catenin (green) and SHH (red) showing the localisation of SHH in cells within the clusters of pituitaries from the ACP murine model at 18.5 dpc. Note the SHH protein on the cell membrane facing the stromal cells (arrows). b Immunohistochemistry reveals the presence of several β-catenin-accumulating cell clusters on a human ACP sample (arrows). c, d In situ hybridisation on the ACP mouse model (c) and human ACP (d) with Shh antisense riboprobes showing the up-regulation of Shh/SHH in some of the β-catenin-accumulating cell clusters (arrows). e, f In situ hybridisation on the ACP mouse model (e) and human ACP (f) with Ptch1 antisense riboprobes. Ptch1, a target of SHH signalling, is expressed throughout the pituitary of the mouse model and in both the palisading cells (arrowheads) and the β-catenin-accumulating cell clusters (arrows) in the human ACP sample. Scale bars a, b, d, f, 50 μm; c, e 150 μm

Fig. 5

Expression analysis by in situ hybridisation of mouse and human ACP. Mouse pituitaries at 18.5 dpc. a–f Expression of Bmp/BMP members 4, 2 and 7 is up-regulated in pre-tumoral mouse pituitaries at 18.5 dpc and human ACP (arrows). In the ACP mouse model pituitary, expression is mainly restricted to cell clusters. In human ACP, expression is up-regulated in cell clusters, but other tumour cells express BMP2 and BMP7. g, h Ctnnb1 and Fgf4 expression in the ACP mouse model pre-tumoral pituitary is up-regulated in cluster cells (arrows). i, j Fgf3/FGF3 expression is also up-regulated in cluster cells in both mouse and human ACP (arrows). k, l Cxcr4 expression is detected in clusters in the ACP mouse model pituitary (arrows), but it is more widely expressed in human ACP. Scale bars a, c, e, g, h, i, k, 150 μm; b, d, f, j, l 50 μm