Tumour compartment transcriptomics demonstrates the activation of inflammatory and odontogenic programmes in human adamantinomatous craniopharyngioma and identifies the MAPK/ERK pathway as a novel therapeutic target

Abstract

Adamantinomatous craniopharyngiomas (ACPs) are clinically challenging tumours, the majority of which have activating mutations in CTNNB1. They are histologically complex, showing cystic and solid components, the latter comprised of different morphological cell types (e.g. β-catenin-accumulating cluster cells and palisading epithelium), surrounded by a florid glial reaction with immune cells. Here, we have carried out RNA sequencing on 18 ACP samples and integrated these data with an existing ACP transcriptomic dataset. No studies so far have examined the patterns of gene expression within the different cellular compartments of the tumour. To achieve this goal, we have combined laser capture microdissection with computational analyses to reveal groups of genes that are associated with either epithelial tumour cells (clusters and palisading epithelium), glial tissue or immune infiltrate. We use these human ACP molecular signatures and RNA-Seq data from two ACP mouse models to reveal that cell clusters are molecularly analogous to the enamel knot, a critical signalling centre controlling normal tooth morphogenesis. Supporting this finding, we show that human cluster cells express high levels of several members of the FGF, TGFB and BMP families of secreted factors, which signal to neighbouring cells as evidenced by immunostaining against the phosphorylated proteins pERK1/2, pSMAD3 and pSMAD1/5/9 in both human and mouse ACP. We reveal that inhibiting the MAPK/ERK pathway with trametinib, a clinically approved MEK inhibitor, results in reduced proliferation and increased apoptosis in explant cultures of human and mouse ACP. Finally, we analyse a prominent molecular signature in the glial reactive tissue to characterise the inflammatory microenvironment and uncover the activation of inflammasomes in human ACP. We validate these results by immunostaining against immune cell markers, cytokine ELISA and proteome analysis in both solid tumour and cystic fluid from ACP patients. Our data support a new molecular paradigm for understanding ACP tumorigenesis as an aberrant mimic of natural tooth development and opens new therapeutic opportunities by revealing the activation of the MAPK/ERK and inflammasome pathways in human ACP.

Keywords: Craniopharyngioma; IL1-β; Inflammasome; MAPK/ERK pathway; Odontogenesis; Paracrine signalling; Trametinib.

Conflict of interest statement

P.B. has received institutional grants from SOBI, Roche and Novartis; consultancy fees from Roche; and lecturing fees from SOBI and Novartis. The other authors declare that they have no conflict of interest.

Figures

Fig. 1

Bioinformatics analysis of gene expression profiling of human ACP whole tumours identifies modules of genes potentially associated with specific tumour cell compartments. a Representative histology of ACP samples showing and areas of tumour (T), reactive glial tissue (G), wet keratin/ghost cells (WK), epithelial whorls (C) (epithelial whorls referred to in this paper as clusters), palisading epithelium (PE) and stellate reticulum (SR). Immunohistochemistry using antibodies against β-catenin (β-cat) on case JA029 showing clusters of cells with nuclear-cytoplasmic accumulation. Scale bars 400 μm (top left panel), 100 μm (other three panels). b Scatterplot revealing a significant correlation between CTNNB1 mutation frequency and histologically assessed tumour content. # case JA011; * case JA005; controls: fetal pituitaries and NFPA tissues. See Suppl. Table 1 (Online Resource 2) for sample details. c Principal component analysis plot showing the separation between adamantinomatous craniopharyngioma (ACP), non-functioning pituitary adenoma (NFPA) and control fetal pituitary tissues (fetal). d Bar plot of selected statistically significant and differentially expressed genes, as assessed by DESeq2, in ACP tumours compared with control fetal tissue. Higher than 0 means higher expression in ACP and lower than 0 means higher in control tissue. The most up-regulated genes in ACP tumours are keratins. Other up-regulated genes include WNTs and WNT pathway targets (e.g. NOTUM, AXIN2, LEF1), genes known to be expressed in ACP (e.g. FGFs, BMPs) and previously suggested therapeutic targets (SHH, MMP12, MMP9, EGFR). Pituitary transcription factors (LHX3, POU1F1) and pituitary hormones (e.g. FSHB, GH1, TSHB) are up-regulated in controls. See Suppl. Table 3 (Online Resource 5) for details. Error bars = 1 standard error, *** adjusted p value < 1 × 10−9. e Multidimensional scaling plot of expression patterns of the 5000 most differentially expressed genes included in the weighted gene co-expression network analysis (WGCNA) analysis. The colour of each gene indicates it membership to a co-expressed gene expression module. f Heatmap of correlations between each module’s gene expression profile and phenotypic information. Scale bar indicate r value − 1 to + 1. For instance, the brown module shows a strong correlation with tumour content and mutational frequency, whilst the blue module correlates with the presence of glial reactive tissue and GFAP

Fig. 2

Gene profiling of laser capture microdissected tumour cells confirms the WGCNA, identifying modules associated with tumour cell compartments and revealing novel ACP genes. a Scheme of the experimental approach. Histological sections of tumour samples JA004 and JA029 were subjected to laser capture microdissection (LCM) to isolate β-catenin-accumulating cell clusters (C), palisading epithelium (PE) and glial reactive tissue (G). Biological duplicates were performed for clusters and palisading epithelium in case JA004. RNA was purified from each of these tumour cell compartments, amplified and sequenced. b Principal component analysis reveals grouping of the data from laser capture microdissected samples. c Gene set enrichment analysis revealing the enrichment of an inflammatory signature in microdissected glial reactive tissue relative to tumour tissue (i.e. genes included in clusters plus palisading epithelium), whilst a WNT signalling expression signature is associated with the microdissected tumour tissue. Enrichment for WNT signalling was stronger in the clusters relative to both PE and glial tissue. d Gene set enrichment analysis showing the enrichment of the brown module genes with a signature of tumour cell compartments (i.e. genes including in cluster cells plus palisading epithelium). In contrast, both the blue and turquoise module genes are predominantly expressed by reactive glia. e Double immunofluorescence staining revealing the expression of BCL11B and TP63 in the epithelial components of the tumour, including palisading epithelium (PE) and β-catenin accumulating clusters (C), but not in reactive glial tissue (G). NES normalised enrichment score, FDR false discovery rate. Scale bars 100 μm

Fig. 3

ACP and developing mammalian tooth share common molecular signatures. a Gene set enrichment plots showing that ACP tumours are enriched for genes expressed by ameloblasts and inner enamel epithelium. Results obtained from using the RNA dataset from profiling whole ACP tumours. b Expression of relevant ameloblast-related genes is significantly expressed at higher levels in whole ACP tumours compared with control tissues (fetal pituitary and NFPA). See Suppl. Table 5 (Online Resource 7) for details (*** adjusted p value < 1 × 10−7, * adjusted p value = 0.028). c Gene set enrichment plots showing that the cluster cells are enriched for genes expressed in the enamel knot whilst palisading epithelium shows a signature of inner enamel epithelia at cap stage. Results obtained from using the RNA dataset from profiling microdissected ACP compartments. d Double immunofluorescent staining reveals the co-expression of p21/CDKN1A and EDAR, two enamel knot markers, in the β-catenin-accumulating clusters. e Gene set enrichment analysis showing that both the inner enamel epithelium and ameloblast gene signatures are enriched in the embryonic mouse ACP model compared with wild-type controls (WT). f Enrichment plots confirming that mouse clusters from both the embryonic and inducible ACP mouse models show a molecular signature of the enamel knot. NES normalised enrichment score, FDR false discovery rate. Scale bars 100 μm

Fig. 4

Identification of the activation of the MAPK/ERK, TGFB and BMP signalling pathways in human ACP. a Immunohistochemistry revealing the expression of phosphorylated ERK1/2 (pERK1/2), a read out of active MAPK/ERK pathway, at the tips of the invading tumour epithelium (palisading epithelium, arrows in a, a‴ and a″″) and within reactive glial tissue (G; arrows in a″). b Double immunofluorescent staining showing pERK1/2 expression in the palisading epithelium (PE) around the β-catenin accumulating clusters (C), which express several activating ligands of the MAPK/ERK pathway (see main text for details). Note that cells within the reactive glial tissue (G) are also pERK1/2 positive. c Double immunofluorescence revealing abundant Ki67+ve cells in the palisading epithelium close to clusters. d Double immunofluorescence showing Ki67 and pERK1/2 co-expression within the palisading epithelium (PE). e Double immunofluorescence showing pSMAD3 staining, indicating activation of TGFβ signalling, in both tumour and reactive glia, with strongest signal in reactive tissue adjacent to tumour epithelia (arrowhead). Double immunofluorescence reveals pSMAD1/5/9 staining, indicating BMP signalling in cells within and adjacent to the β-catenin-accumulating clusters (C). Note the absence of staining in the palisading epithelium (PE). Scale bars: a–a″″ 200 μm; b–f 100 μm

Fig. 5

Identification of the activation of the MAPK/ERK, TGFB and BMP signalling pathways in the ACP embryonic mouse model. Double immunofluorescent staining on histological sections of neoplastic pituitaries of the ACP embryonic mouse model at postnatal day 1 (P1). Note the widespread expression of pERK1/2 in cells around the β-catenin-accumulating cell clusters, which show no expression of this marker. pSMAD3 and pSMAD1/5/9 staining is also predominant in cells surrounding the clusters, but occasionally weak staining is observed in some cluster cells (arrowheads). Scale bars 50 μm

Fig. 6

Ex vivo inhibition of the MAPK/ERK pathway in mouse ACP results in decreased proliferation and increased apoptosis of tumour cells. Neoplastic pituitaries of the ACP embryonic mouse model were cultured in the presence of the MEK inhibitor trametinib (2 or 20 nM) or the vehicle control (DMSO) for 18 h. Following histological processing, sections were immunostained against β-catenin and pERK1/2 (readout of active MAPK/ERK pathway; a), Ki-67 (proliferation marker; b) and cleaved caspase-3 (apoptosis marker; c). Quantitative analysis showing a significant dose-dependent reduction in Ki-67 proliferative index (d; 20 nM) and an increase in apoptosis (e; 2 and 20 nM) in trametinib-treated relative to vehicle-treated control. Kruskal–Wallis with Dunn’s post-test **p < 0.01; ***p < 0.001. Mean of 4.1 × 103 nuclei for each point. Scale bars 50 μm

Fig. 7

Ex vivo inhibition of the MAPK/ERK pathway in human ACP results in decreased proliferation and increased apoptosis of tumour cells. Small pieces of three human ACP tumours were cultured in the presence of the MEK inhibitor trametinib (2 or 20 nM) or the vehicle control (DMSO) for 18 h. Following histological processing, sections were immunostained against β-catenin and pERK1/2 (readout of active MAPK/ERK pathway; a), Ki-67 (proliferation marker; b) and cleaved caspase-3 (apoptosis marker; c). Quantitative analysis showing a significant dose-dependent reduction in Ki-67 proliferative index (d; 2 and 20 nM) and an increase in apoptosis (e; 20 nM) in trametinib-treated relative to vehicle-treated control. Kruskal–Wallis with Dunn’s post-test **p < 0.01; ***p < 0.001. Mean of 1.6 × 104 nuclei for each point. Scale bars 50 μm

Fig. 8

Characterisation of the immune microenvironment in the solid component and cystic fluid of human ACP. a Immunohistochemistry showing infiltration of myeloid (CD68+ve) and lymphoid (CD3+ve) within human ACP tumour (T) and reactive glial tissue (G). CD68+ve and IBA1+ve immunostaining is observed in close association with the cholesterol clefts (arrows). Likewise, immunohistochemistry against the chemokine CCL2 is detected near the cholesterol clefts. b The expression of the cytokines IL18, IL1B and IL10 correlate significantly with CD14 expression, a marker preferentially expressed in monocytes/macrophages, in the 24 samples (ACP tumours and control tissues) profiled by RNA-Seq. c Multiplex ELISA quantification of cytokine protein expression within the solid tumour (n = 8 tumours; left) and cystic fluid (n = 10 samples; right). IL8, IL18, IL6 and IL1B are the highest expressed cytokines in the solid tumour. In the cystic fluid, levels of IL6 and IL8 are the highest, but all the other cytokines are also detected. For solid tumour values were normalised against total protein and for cystic fluid samples against volume. The blue dots represent the value obtained of each cytokine for each sample and the red dots represent the median. Scale bars 100 μm

Fig. 9

Activation of the inflammasomes underlies the ACP inflammatory response. a Gene set enrichment plots showing that human ACP tumours are enriched for genes expressed by macrophages, chondrocytes and uterine muscle cells exposed to IL1B in culture conditions. b Gene set enrichment plots reveal a molecular signature of atherosclerotic plaques in human ACP. Results are based on data obtained from RNA-Seq profiles from whole ACP tumours and control tissues (fetal pituitaries and NFPA). c The levels of IL6, IL8 and TNFα protein correlate with levels of IL1B in human ACP cystic fluid (n = 10 cystic fluid samples; determined by ELISA). NES normalised enrichment score, FDR false discovery rate

Fig. 10

Schematic summary of the findings. Molecular and histological relationships between ACP pathogenesis and tooth development. The enamel knot and the β-catenin-accumulating clusters, which both have similar expression profiles and comparable histology, act as signalling hubs through the secretion of a several growth factors acting in an autocrine and/or paracrine manner on the surrounding cells, i.e. the enamel epithelium/dental mesenchyme in the forming tooth or the palisading epithelium, stellate reticulum and glial reactive tissue in ACP. Reciprocal signalling from surrounding tissues to the enamel knot and clusters is indicated by double-headed arrows. In the glial reactive tissue, cholesterol crystals activate the inflammasomes resulting in the secretion of IL1B, which in turn acts on the local immune effector cells to drive an inflammatory response