Increased Wingless (Wnt) signaling in pituitary progenitor/stem cells gives rise to pituitary tumors in mice and humans

Abstract

Wingless (Wnt)/β-catenin signaling plays an essential role during normal development, is a critical regulator of stem cells, and has been associated with cancer in many tissues. Here we demonstrate that genetic expression of a degradation-resistant mutant form of β-catenin in early Rathke's pouch (RP) progenitors leads to pituitary hyperplasia and severe disruption of the pituitary-specific transcription factor 1-lineage differentiation resulting in extreme growth retardation and hypopituitarism. Mutant mice mostly die perinatally, but those that survive weaning develop lethal pituitary tumors, which closely resemble human adamantinomatous craniopharyngioma, an epithelial tumor associated with mutations in the human β-catenin gene. The tumorigenic effect of mutant β-catenin is observed only when expressed in undifferentiated RP progenitors, but tumors do not form when committed or differentiated cells are targeted to express this protein. Analysis of affected pituitaries indicates that expression of mutant β-catenin leads to a significant increase in the total numbers of pituitary progenitor/stem cells as well as in their proliferation potential. Our findings provide insights into the role of the Wnt pathway in normal pituitary development and demonstrate a causative role for mutated β-catenin in an undifferentiated RP progenitor in the genesis of murine and human craniopharyngioma.

Fig. 1.

Nuclear accumulation of β-catenin and activation of the Wnt pathway occurs in a small subset of anterior pituitary progenitors. (A) Histological sections of Hesx1^Cre/+;Ctnnb1^+/lox(ex3) pituitary glands from 9.5–15.5 dpc immunostained with a specific β-catenin antibody (Table S2) and counterstained with hematoxylin. Magnified views of the boxed regions in the upper row are shown in the lower row. Note that accumulation of β-catenin begins in a few cells within RP epithelium (arrowheads). (B) β-Catenin immunofluorescent detection in the postnatal pituitary at postnatal day 11 (P11) showing the persistence of β-catenin–accumulating cell clusters (arrowheads). (C) The spotted X-gal staining in the Hesx1^Cre/+;Ctnnb1^+/lox(ex3);BAT-gal pituitary indicates that expression of beta-galactosidase occurs only in cell clusters. The posterior lobe (pl) is strongly stained in both genotypes. (D) Quantitative RT-PCR analysis of dissected pituitaries at 15.5 dpc showing a significant increase in the expression of the canonical Wnt/β-catenin targets Lef1, Axin2, and Cyclin D1, but not of paired-like homeodomain transcription factor 2 (Pitx2), C-myc, and Cyclin D2 in the heterozygous pituitaries relative to controls (n = 5–9), (*P < 0.01, **P < 0.001, t test). (E) In situ hybridization with Axin2 and Lef1 and Cyclin D2 immunofluorescence on 15.5-dpc pituitary glands showing clusters of higher expression in the double-heterozygous pituitary (arrowheads, Right); these clusters are absent in the control (Left). (F) Hesx1^Cre/+;Ctnnb1^+/lox(ex3);R26^YFP/+ triple-heterozygous pituitary at 15.5 dpc showing widespread expression of YFP throughout most of the pituitary but focal accumulation of intracellular β-catenin only in cell clusters (arrowheads). A magnified view of the boxed region in the Merge image is shown on the right. (Scale bars: 100 μm.)

Fig. 2.

Hesx1^Cre/+;Ctnnb1^+/lox(ex3) mice develop hypopituitarism and pituitary tumors that resemble human ACP. (A) A 5-wk-old heterozygous mutant exhibits marked dwarfism relative to a control same-sex littermate. (B) Growth hormone (GH) content in the pituitary gland is reduced significantly in Hesx1^Cre/+;Ctnnb1^+/lox(ex3) heterozygous mutants compared with Ctnnb1^+/lox(ex3) control littermates (Left), but ACTH levels are comparable between genotypes (Right). Data shown are mean ± SEM of five pituitaries. (P < 0.001, t test). (C) The pituitary gland (arrowheads, Upper) is not distinguishable in a Hesx1^Cre/+;Ctnnb1^+/lox(ex3) double heterozygote, and there is a large tumor in this location (arrows, Lower). (D) Control pituitary (Upper) and a pituitary tumor (Lower) stained with H&E showing the presence of large cysts (arrowheads) and blood (arrows). al, anterior lobe; il, intermediate lobe; pl, posterior lobe. (E) Log-rank (Mantel–Cox) survival test of Ctnnb1^+/lox(ex3) control and Hesx1^Cre/+;Ctnnb1^+/lox(ex3) heterozygous mutants that survived weaning (n = 16 animals per group). Median survival time is 11 wk for double-heterozygous mice (P < 0.0001). (F) H&E staining of histological sections of human ACP (Right) and murine tumor (Left) showing the presence of microcystic change (stellate reticulum; arrows) and nodular structures (cell clusters; arrowheads) in both. (G) In situ hybridization for Gh reveals no expression within the Hesx1^Cre/+;Ctnnb1^+/lox(ex3) tumor, but Gh-expressing cells are detected in what remains of normal pituitary tissue at the periphery of the tumor. (H) β-Catenin is detected in the cytoplasmic membrane of control pituitaries (arrows; arrowheads indicate nonspecific signal in the capillaries; Upper), but it is intracellular in many cells of the Hesx1^Cre/+; Ctnnb1^+/lox(ex3) tumor (arrows; Lower). (I) β-Catenin and Ki67 immunofluorescence detection in human ACP (Lower) and 18.5-dpc double-heterozygous pituitary (Upper) showing accumulation of β-catenin in cell clusters, which are Ki67-negative. (Scale bars: 100 μm in D and G; 500 μm in F and H; 50 μm in I.)

Fig. 3.

β-Catenin–accumulating cells show phenotypic features of pituitary progenitor/stem cells. (A) Double immunostaining for β-catenin (green) and specific markers (red) in Ctnnb1^+/lox(ex3) controls and Hesx1^Cre/+;Ctnnb1^+/lox(ex3) double-heterozygous pituitaries at 18.5 dpc. BrdU incorporation short pulse reveals abundant cells in S-phase of the cell cycle throughout the gland but very few BrdU-positive cells in the clusters. Cyclin D1 and Cyclin D2, markers of undifferentiated pituitary progenitors, are expressed within the cell clusters. Overall expression of p57^kip2, which is transiently expressed in cells that are between the proliferation and differentiation states, is increased in the Hesx1^Cre/+;Ctnnb1^+/lox(ex3) anterior pituitary but is not expressed in β-catenin–accumulating cells. SOX2-expressing cells concentrate around the lumen of the gland, which contains pituitary progenitor/stem cells, and this region is thickened in the Hesx1^Cre/+;Ctnnb1^+/lox(ex3) pituitary. Note that expression of SOX2 is detected in some but not all β-catenin–accumulating cell clusters. Expression of SOX9 is rare within the cell clusters, but there are abundant SOX9-positive cells surrounding the β-catenin–accumulating cell clusters and around the lumen. Expression of the stem cell marker p27^kip1 is up-regulated in the clusters. (B) In human ACP, SOX9 immunofluorescence is widespread throughout the tumor, but β-catenin–accumulating clusters are negative. al, anterior lobe; il, intermediate lobe; pl, posterior lobe. (Scale bars: 50 μm.)

Fig. 4.

Increased numbers of pituitary progenitor/stem cells and an elevated proliferation rate underlie the formation of the murine tumors. (A) Total numbers of progenitor/stem cells (colony-forming cells) are significantly elevated in the Hesx1^Cre/+;Ctnnb1^+/lox(ex3) pituitaries relative to controls from late gestation to adulthood (n = 5–8 pituitaries per stage). (B) Time-lapse microscopy frames showing the formation of a colony from a single progenitor/stem cell (arrowhead, Upper left). (C) Time-lapse microscopy showing that the proliferation rate of pituitary progenitor/stem cells is increased in Hesx1^Cre/+;Ctnnb1^+/lox(ex3) mutant (Lower row) relative to Ctnnb1^+/lox(ex3) control pituitaries (Upper row). (D) Representative examples of lineage trees indicating faster division times in the double heterozygotes. (E) Photographs of tissue culture plates containing fixed and hematoxylin-stained colonies demonstrate the presence of higher numbers and overall larger colonies in the Hesx1^Cre/+;Ctnnb1^+/lox(ex3) pituitaries (Right) relative to Ctnnb1^+/lox(ex3) controls (Left). (F) Expression of mutant β-catenin in committed progenitors (Pit1-Cre;Ctnnb1^+/lox(ex3)) (Left) or terminally differentiated hormone-producing cells (Gh-Cre;Ctnnb1^+/lox(ex3) (Center) and Prl-Cre;Ctnnb1^+/lox(ex3)) (Right) is not tumorigenic, and pituitaries from adult mice are normal. (Scale bars: 100 μm.)