TheWnt Signalling Cascade and the Adherens Junction Complexin Craniopharyngioma Tumorigenesis

Veronica Preda & Sarah J. Larkin & Niki Karavitaki &Olaf Ansorge & Ashley B. Grossman

# Springer Science+Business Media New York 2014

Abstract Craniopharyngiomas are epithelial, sellar tumourswith adamantinomatous (aCP) and papillary (pCP) subtypes.The aCP type usually occurs during childhood and pCP inmiddle-aged adults; aCPs often contain mutations inCTNNB1, encoding β-catenin, a component of the adherensjunction and a mediator ofWnt signalling. No suchmutationalevent has been associated with pCPs, where the BRAF geneappears to be more important. In a large series of 95craniopharyngiomas, we show that the aCP subtype harboursmutations in CTNNB1 in 52 % of cases, while the pCPsubtype does not, with agreement between immunohisto-chemistry and sequencing methods in the majority of cases.When present, the CTNNB1mutation is found throughout theaCP tumour, while translocation of β-catenin from membraneto cytosol and nucleus is restricted to small cell clusters nearthe invading tumour front. We observed translocated β-catenin in 100 % of aCPs, occurring not only in cell clustersbut also in individual cells scattered throughout the tumour.We characterised the adherens junction involving α-catenin,β-catenin, γ-catenin, p120 and E-cadherin (cytosolic andmembranous components). Although suggested to be

important in other sellar mass tumourigenesis pathways, therewas no disruption of the adherens junction in these tumours,indicating that a loss of junctional integrity is not associatedwith β-catenin translocation or mutation. We conclude thatmutations inCTNNB1 underlie tumourigenesis in the majorityof aCPs, which are distinct morphologically and at the molec-ular level from pCPs.

Keywords Craniopharyngioma .β-Catenin .

Adamantinomatous . Adherens junction . Papillary

Introduction

Craniopharyngiomas (CPs) are complex, epithelial tumours ofthe sellar region that occur with an incidence of 1.3 per millionperson years. No gender differences are observed, but age atdiagnosis shows a bimodal distribution with peaks at 5–14and 65–74 years [1]. Two subtypes of tumour have beenrecognised for over 100 years (reviewed in [2]):adamantinomatous craniopharyngiomas (aCP) occur predom-inant ly in chi ldhood, whi le the rarer papi l la rycraniopharyngioma (pCP) is seen almost exclusively in adults(ratio aCP/pCP cases 9:1). Surgical treatment is usually theoptimal first-line therapy; however, this is challenging due totheir frequently large size and irregular margins and the ad-herence of the tumour to vital surrounding structures such asthe visual pathways and the hypothalamus. As a result, com-plete surgical removal is often not possible and indeed may beinadv i s ab l e , and pa r t i a l r emova l— i f po s s ib l etranssphenoidally—followed by external beam radiotherapyis usually recommended [3–5].

Studies of the pathogenesis of aCPs have highlighted therole of the Wnt pathway mediator and adherens junctioncomponent β-catenin [6]. Mutations that ablate critical serineand threonine residues (S33, S37, T41 and S45) of β-catenin

S. J. Larkin :O. AnsorgeNuffield Department of Clinical Neurosciences, John RadcliffeHospital, Headley Way, Oxford OX3 9DU, UK

S. J. Larkin :O. AnsorgeDepartment of Neuropathology, John Radcliffe Hospital, HeadleyWay, Oxford OX3 9DU, UK

V. Preda (*) :N. Karavitaki :A. B. GrossmanDepartment of Endocrinology, Oxford Centre for Diabetes,Endocrinology and Metabolism, Churchill Hospital, Old Rd,Headington, Oxford OX3 7LE, UKe-mail: vpre3773@uni.sydney.edu.au

V. PredaKolling Institute, Royal North Shore Hospital, University of Sydney,Sydney, Australia

Endocr PatholDOI 10.1007/s12022-014-9341-8

allow it to evade phosphorylation by the destruction complexand escape ubiquitin-mediated proteasomal degradation([7–16] and reviewed in [17]). Consequently, β-catenin accu-mulates in the cytosol and nucleus [18], switching on Wntsignalling and mediating transcription of Wnt target genes inthe absence of a Wnt ligand. Analysis of CTNNB1, the genethat encodes β-catenin, in craniopharyngiomas has revealedmutations at S33, S37, T41 or S45 in aCPs at a frequency ofaround 60 % (ranging from 16 to 100 %). No mutations havebeen reported in pCPs [6, 19–24]. In contrast, cytosolic andnuclear accumulation of β-catenin has been reported in 93 %of aCPs (range 78–100 %) but never in pCPs [6, 20, 21, 23,25–28]. Expression of mutated β-catenin in the early stages ofmouse pituitary organogenesis leads to aberrant pituitary de-velopment and the appearance of tumours resembling thehuman aCP, suggesting that β-catenin mutation could be atumourigenic event [29, 30]. However, the presence of amutation inCTNNB1 does not lead to β-catenin accumulationthroughout the whole tumour [20, 21, 23, 31], and so, therelationship between mutation at β-catenin phosphorylationsites and its accumulation in the nucleus and cytosol remainsto be clarified. Conversely, BRAF mutations have recentlybeen associated with pCPs [32] [24].

In addition to its role in Wnt signalling, β-catenin is also acomponent of the adherens junction complex and facilitatescell-cell adhesion, along with α-catenin, γ-catenin and p120,by tethering the actin cytoskeleton to E-cadherin [33, 34].Previous studies have demonstrated that the cells in aCPs withcytosolic and nuclear accumulation of β-catenin display in-creased motility compared with surrounding cells, a propertythat was reduced by siRNA-mediated knockdown ofCTNNB1 [28]. Down-regulation of the expression of compo-nents of the adherens junction, including E-cadherin and p120resulting from mutation, epigenetic silencing and allelic loss,has been implicated in the pathogenesis of several humanmalignancies including breast [35–37], pancreatic [38], gas-tric [39] [40], and colorectal cancers [41], and disruption ofthis complex is a common tumourigenic event.

Given the role of adherens junction disassembly in cellmigration and tumour progression, we examined the in-tegrity of this complex in both aCPs and pCPs to deter-mine whether the translocation of β-catenin seen in aCPsmight be associated with adherens junction disruption andwhether mislocalisation of other members of the complexmight be associated with tumourigenesis in pCPs. Wehave therefore examined the relationship between muta-tion in CTNNB1 and the subcellular location of β-cateninin a large series of 95 craniopharyngiomas. The frequentsites of phosphorylation in exon 3 of CTNNB1 weresequenced, and the subcellular location of β-catenin andother components of the adherens junction complex (E-cadherin, α-catenin, γ-catenin and p120 catenin) weredetermined in both aCPs and pCPs.

Materials and Methods

We retrieved 131 specimens carrying a neuropathologicaldiagnosis of craniopharyngioma from the surgical neuropa-thology archive of the Oxford Brain Bank. Diagnostic reviewconfirmed classical features of aCP (defined by the presenceof wet keratin nodules, calcification and/or stellate reticulumsurrounded by peripheral palisaded epithelium) and pCP (de-fined by the absence of wet keratin nodules, calcification orstellate reticulum, and the presence of squamous epitheliumwith fibrovascular cores). Cases with insufficient epitheliumfor a definitive morphological diagnosis and ‘redo’ surgicalspecimens on the same patient were excluded, totalling 36specimens. Studies were conducted under multi-site and localREC approval.

Immunohistochemistry

Immunohistochemical analysis was performed on 4-μm sec-tions from formalin-fixed paraffin-embedded (FFPE) tumourspecimens. Following dewaxing through graded alcohols,endogenous peroxidase activity was blocked (3 % (v/v)H2O2 in phosphate-buffered saline (PBS), pH 7.3, 20 minwith orbital shaking). Epitope retrieval was achieved byautoclaving in sodium citrate (10 mM, pH 6.0, 10 min) (β-catenin). Sections were blocked with serum block (HiStar3000 kit, AbD Serotec (Oxford, UK)) for 15 min and thenincubated overnight at 4 °C with primary antibody diluted inPBS (see Table 1 for antibodies, concentrations and suppliers).All subsequent steps were carried out according to the manu-facturer’s instructions (AbD Serotec) with the following mod-ifications: HRP polymer was applied for 40 min and DAB for5 min. Sections were counterstained with Cole’shaematoxylin.

Of note, ECAD clone 4A2C7 was used, which is specificand does not stain the nuclei of normal human cortex. Previ-ously published pituitary work has used the non-specificECAD clone 36, resulting in false interpretation of transloca-tion of ECAD and its role in Wnt signalling in pituitarytumours [42]. Others have also demonstrated non-specificityof this ECAD clone 36 for immunohistochemistry on FFPEsections [43].

Sequencing of CTNNB1 exon 3

DNA was extracted from 5×10-μm sections of FFPE tissuefrom archival surgical specimens (QiaAmp FFPE DNA kit,Qiagen (Crawley, UK)). PCR was performed to generate a269-bp amplicon including codons S33, S37, T41 and S45 inexon 3 of CTNNB1. Primers were as follows: sense (5′-GATTTGATGGAGTTGGACATGG-3′) and antisense (5′-TGTTCTTGAGTGAAGGACTGAG-3′). DNA template (50 ng) wasadded to 10× PCR buffer solution (10 % v/v, Qiagen), MgCl2

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(final concentration 4 mM, Qiagen), dNTPs (200 μM)(Promega, Southampton, UK), 0.5 U of Taq polymerase(HotStarTaq Plus, Qiagen) and 200 nM each of sense andantisense primers in a total volume of 20 μl. Cycling condi-tions were as follows: 95 °C for 5 min, followed by 35 cyclesof 95 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min. DNAtemplate (100 ng) was added to 10× PCR buffer solution(10 % v/v, Qiagen), MgCl2 (final concentration 4 mM,Qiagen), dNTPs (200 μM) (Promega, Southampton, UK),0.5 U of Taq polymerase (HotStarTaq Plus, Qiagen) and400 nM each of sense and antisense primers in a total volumeof 20 μl. Cycling conditions were as follows: 95 °C for 5 min,followed by 35 cycles of 95 °C for 30 s, 60 °C for 30 s and72 °C for 40 s, and a final elongation step at 72 °C for 1 min.Products were examined by agarose gel separation and puri-fied (MinElute PCR Purification kit (Qiagen)). Sequencingreactions were performed using BigDye Terminator chemistryand an ABI-3730 sequencer.

Results

Nuclear Translocation of β-Catenin Is a Feature of aCPsbut Not pCPs

All the aCP cases in our series for which there was a sufficientepithelium to make a diagnosis (N=72) contained cells thatshowed nuclear and cytosolic accumulation of β-catenin(Fig. 1a). This pattern was absent in all of the pCP cases(N=23; Fig. 1b).

Accumulation of β-catenin has previously been reportedonly in whorls of epithelial cells that form near to the infiltrat-ing edge of the tumour. However, in our series, we observedthat translocation of β-catenin did not always coincide withthese whorl formations and could also occur in individual,isolated cells (Fig. 2).

Translocation of β-Catenin Is Mutation-Independent and NotAssociated with Disruption of the Adherens JunctionComplex

Examination of the adherens junction complex in this seriesrevealed that β-catenin is the only member of this complex toundergo translocation and accumulate in the cytosol and nu-cleus. No difference in the localisation of E-cadherin, α-ca-tenin, γ-catenin or p120 catenin was observed in any of theaCPs or pCPs (Fig. 3). This was true for both the whole slideand for individual scattered cells showing β-catenin translo-cation. In aCP cases, the pattern of β-catenin staining wascompared to mutations in CTNNB1. Cases were classifiedaccording to the presence of epithelial whorls and the patternof β-catenin translocation as (1) individual-cell pattern of β-catenin accumulation, (2) whorl pattern of β-catenin accumu-lation or (3) whorl pattern without β-catenin accumulation.No relationship was found between CTNNB1 mutation andthe pattern of β-catenin translocation (Fisher’s exact test, P=0.182, 0.330 and 0.279, respectively).

Sequencing of the region of exon 3 of CTNNB1 containingS33, S37, T41 and S45 was possible in 42 specimens, 9 ofwhich were pCPs (the quality of the DNAwas insufficient inthe remainder of cases). Mutation was found in 52 % of aCPsin this series but in none of the pCPs (Table 2).

Discussion

Accumulation of β-Catenin Occurs in All aCPs in Clustersor Individual Cells and Is Unrelated to CTNNB1 Mutationin Bulk Tumour

In all of the cases of aCP in our series for which sufficientepithelium was available to make a definitive morphologicaldiagnosis, accumulation of β-catenin was observed in eitherclusters of cells or individual epithelial cells. This finding

Table 1 Antibody, concentration, suppliers and dilution factor for immunohistochemistry

Antibody Species Manufacturer Dilution

E-cadherin (membrane) Mouse monoclonal (clone 36B5) Leica 1:50

E-cadherin (cytosol) Mouse monoclonal (clone 4A2C7) Invitrogen 1:800

α-Catenin Mouse monoclonal (clone 5) BD Transduction Laboratories 1:500

β-Catenin Rabbit monoclonal (clone E247) Epitomics 1:1,000

γ-Catenin Mouse monoclonal (clone 15) BD Transduction Laboratories 1:1,000

p120 Catenin Rabbit monoclonal (clone EPR357(2)) Epitomics 1:1,000

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a b

_____

a

b

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confirms the utility of β-catenin translocation as a tool in thediagnosis of this tumour subtype; the absence of observableβ-catenin translocation is suggestive of a pCP. Previous stud-ies have reported lower frequencies of observed β-catenintranslocation [6, 20, 21, 25–28, 44]; some of these studiesdo not report individual cells with cytosolic or nuclear β-catenin which may have been present, and so, the number ofcases with β-catenin translocation may have beenunderestimated. Accumulation of β-catenin in the cytosoland nucleus of individual epithelial cells has been reportedpreviously [21, 45]. Cells with β-catenin translocation havebeen described adjacent to ghost cells [21, 27, 46], and Katoet al. proposed that cells with β-catenin translocation repre-sent a transitional form between epithelial and ghost cells.Sequencing of bulk tumour revealed no relationship betweenmutations at S33, S37, T41 or S45 ofCTNNB1 and the patternof cytoplasmic or nuclear accumulation of β-catenin. Thefinding that not all aCPs harbour a mutation in CTNNB1 atthese sites suggests that, while mutation at these sites may be atumourigenic event in this lesion, it is neither sufficient nornecessary for tumour formation. This appears to be in contrastto studies of the effect of CTNNB1mutation on early pituitaryformation in mice, which showed that the expression of mu-tated β-catenin under the control of transcription factor Hesx1(required for the formation of Rathke’s pouch) led to the

development of lesions containing clusters of cells with cyto-solic and nuclear β-catenin reminiscent of those found inhuman aCPs [30], strongly implying a role for β-cateninmutation in the development of aCP. It should be noted thatour study did not search for mutations at loci other than thefour conserved phosphorylation sites inCTNNB1which couldhave led to an underestimation of the proportion of specimensthat harbour CTNNB1 mutations enabling stabilisation of β-catenin, for example mutations at codon 32 [6, 20–23]).However, in studies that did examine mutations at other lociby Sanger dideoxy sequencing methods, the rate of CTNNB1mutationwas still not 100%, [6, 20–23], suggesting that eitherthis method is not sufficiently sensitive to detect the presenceof a mutation in all samples [47, 48] or there may be anothertumourigenic event underlying formation of this tumour sub-type. The use of FFPE tissue for the study allows directcomparison of findings to previous studies that report thefrequency of CTNNB1 mutations [6, 20, 49]. The use offrozen material, along with next-generation sequencing tech-nology, would yield much more robust results and with suffi-cient read depth would increase the sensitivity of the sequenc-ing data in cases with a low ratio of tumour epithelium tonormal tissue. In our series, the majority of mutations inCTNNB1 were at T41 (88 % of mutations) and all wereT41I. This is in contrast to other published studies that reporta more even distribution of mutations with the majority ofmutations at S33 (from 33 to 60% [6, 20–23]). The reason forthe difference in distribution of mutations at the four con-served phosphorylation residues is unclear. Phosphorylationof β-catenin occurs at S45 (by CK1), T41, S37 and S33 (byGSK3β) sequentially [8], and phosphorylation of S33 is nec-essary for subsequent ubiquitination ofβ-catenin, so mutationat any of these loci should be sufficient to producedegradation-resistant β-catenin. It therefore seems unlikelythat the difference in the site of mutation affects thestabilisation of β-catenin.

The confinement of β-catenin accumulation to small clus-ters of cells or individual cells despite the presence of a

�Fig. 1 a Adamantinomatous craniopharyngioma. Classical featuresinclude a peripheral palisading epithelium (open arrow), surrounding aloose stellate reticulum (SR) containing nodules of ‘wet keratin’ or ‘ghostcells’ (GC). aWhorls of epithelial cells near the infiltrating tumour edge(solid arrow) may also be visible. Translocation of β-catenin (brownreaction product, c) may be visible in clusters of cells associated withepithelial whorls (arrows, b and c). Haematoxylin and eosin (a, b). β-catenin immunohistochemistry (brown reaction product c). Scale bars a80 μm, c 100 μm. b Papillary craniopharyngioma. Characterised byclefted (pseudo-)papillae and lacking the stellate reticulum, ghost cellsand epithelial whorls characteristic of the adamantinomatous type. β-Catenin remains at the membrane throughout these tumours. aHaematoxylin and eosin. b β-catenin immunohistochemistry (brownreaction product). Scale bar 100 μm

Fig. 2 Relationship betweenepithelial whorl formations andβ-catenin translocation.Characteristic whorls of epithelialcells may (open arrows a, b) ormay not (solid arrows a, b)correspond to translocation of β-catenin from membrane tocytosol/nucleus. Translocation ofβ-catenin may also occur inisolated cells not associated withepithelial whorls (a, b). Scalebar=80 μm

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stabilising CTNNB1 mutation in bulk tumour has not beensatisfactorily explained. Other authors have observed thatthese clusters show pituitary stem cell-like characteristics[29, 45, 50–52] and have lower proliferation indices comparedto surrounding epithelium as assessed by Ki-67 immunohis-tochemistry [21, 25]. These cells also express Sonic hedgehog(SHH), transforming growth factor (TGF) and fibroblastgrowth factor (FGF) at higher levels than surrounding tumourcells, leading some authors to suggest that they represent a‘signalling hub’, providing growth signals to the surroundingtumour [53–55]. In a small numbers of cases, analysis of theCTNNB1 sequence in DNA extracted from microdissected

cells with β-catenin translocation showed differences com-pared to the sequence of CTNNB1 from bulk tumour [20, 21,23, 31]. A larger series is needed to clarify whether mutationin CTNNB1 may be confined to or different in cells thatspecifically translocate β-catenin.

Translocation of β-Catenin Is Not Associated with AdherensJunction Disruption in aCPs

We characterised the adherens junction involving α-catenin,β-catenin, γ-catenin, p120 and E-cadherin (cytosolic andmembranous components). Previous studies have implicated

Fig. 3 Components of theadherens junction in aCP.Translocation of β-catenin frommembrane to cytosol/nucleusoccurs in clusters of cells near theinfiltrating tumour edge (arrow,a). This translocation is notassociated with disruption of theadherens junction in these cells.α-Catenin (b),γ-catenin (c), p120catenin (d), and E-cadherinextracellular domain (e) andintracellular domain (f) retaintheir membranous location. Scalebar=100 μm

Table 2 Mutation loci of CTNNB1

Exon 3 of CTNNB1 encoding for B-catenin

Residue S33 S37 T41 S45 IHC (translocation of B-catenin) and pattern

aCP sequencing possible in 33specimens

2 (1×S>C, 1×S>F)

0 15 (T>I)

0 100 %

CTNNB1 mutation status and pattern ofstaining

Fisher’s exact test pvalue

Individual cells (4) 0.182

Whorls (13) 0.330

Whorls (0) without 0.279

pCP sequencing possible in ninespecimens

0 0 0 0 0 (0 %)

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the adherens junction in other sellar mass tumourigenesispathways, such as pituitary adenoma pathogenesis [42]. Usinga specific antibody for each of the adherens junction compo-nents, we found that the complex was not disrupted in β-catenin-accumulating cells versus bulk tumour, a novel find-ing in craniopharyngiomas. E-cadherin (both extracellular andintracellular domains),α-catenin, γ-catenin (plakoglobin) andp120 catenin remained at the membrane in all tumour cells.Translocation of β-catenin in clusters or isolated cells istherefore not likely to be attributable to disruption of theadherens junction complex. In this series, β-catenin translo-cation did not always coincide with epithelial whorls of cellsnear to the invading tumour edge, and cells with cytosolic andnuclear β-catenin were often found at the edge of theseformations or in isolated cells (Fig. 1a). Although oftencolocalised, epithelial whorls are not a marker for β-catenintranslocation, and the relationship between these features re-mains to be defined. Inhibition of theWnt pathway has provedchallenging, and effective inhibitors are still largely in devel-opment [56, 57], but our findings clarify the need to exploreother tumourigenesis pathways in order to find novel targetedagents.

Conclusions

In our series, cytosolic or nuclear accumulation of β-cateninwas present in 100 % of aCP cases in either clusters of cells orindividual cells but was never present in pCPs. Mutation inCTNNB1 (S33, S37, T41 or S45) was present in 52 % of aCPsbut was not linked toβ-catenin translocation. Translocation ofβ-catenin from membrane to cytosol or nucleus is not associ-atedwith altered localisation of other members of the adherensjunction complex.

Acknowledgments We acknowledge the Oxford Brain Bank, support-ed by the Medical Research Council (MRC), Brains for Dementia Re-search (BDR) and the NIHR Oxford Biomedical Research Centre. Theresearch was funded by the National Institute for Health Research (NIHR)Oxford Biomedical Research Centre based at Oxford University Hospi-tals NHS Trust and University of Oxford. The views expressed are thoseof the author(s) and not necessarily those of the NHS, the NIHR or theDepartment of Health.

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