Disruption of the Wnt-antagonist Apc in the pituitary stem cells drives the development of adamantinomatous craniopharyngioma

Disruption of the Wnt-antagonist Apc in the pituitary stem cells drives the development of adamantinomatous craniopharyngioma | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Disruption of the Wnt-antagonist Apc in the pituitary stem cells drives the development of adamantinomatous craniopharyngioma James Blackburn , Laura Gomez-Corral , James Nicholson , Ashutosh Rai , Jingyi Xue , Rachael Tan , Kalyana C. Gangaraju , Angelica Gualtieri , Charlotte Hall , Pinaki Dutta , Márta Korbonits , Federico Roncaroli , Shannon W Davis , View ORCID Profile Carles Gaston-Massuet doi: https://doi.org/10.1101/2025.09.28.679021 James Blackburn 1 Centre for Endocrinology, William Harvey Research Institute, Barts & the London School of Medicine and Dentistry, Queen Mary University of London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Laura Gomez-Corral 1 Centre for Endocrinology, William Harvey Research Institute, Barts & the London School of Medicine and Dentistry, Queen Mary University of London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site James Nicholson 1 Centre for Endocrinology, William Harvey Research Institute, Barts & the London School of Medicine and Dentistry, Queen Mary University of London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ashutosh Rai 1 Centre for Endocrinology, William Harvey Research Institute, Barts & the London School of Medicine and Dentistry, Queen Mary University of London , London, UK 2 Department of Biochemistry, University of Punjab , India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jingyi Xue 1 Centre for Endocrinology, William Harvey Research Institute, Barts & the London School of Medicine and Dentistry, Queen Mary University of London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rachael Tan 1 Centre for Endocrinology, William Harvey Research Institute, Barts & the London School of Medicine and Dentistry, Queen Mary University of London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kalyana C. Gangaraju 1 Centre for Endocrinology, William Harvey Research Institute, Barts & the London School of Medicine and Dentistry, Queen Mary University of London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Angelica Gualtieri 1 Centre for Endocrinology, William Harvey Research Institute, Barts & the London School of Medicine and Dentistry, Queen Mary University of London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Charlotte Hall 1 Centre for Endocrinology, William Harvey Research Institute, Barts & the London School of Medicine and Dentistry, Queen Mary University of London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Pinaki Dutta 3 Department of Endocrinology, Postgraduate Institute of Medical Education and Research , Chandigarh, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Márta Korbonits 1 Centre for Endocrinology, William Harvey Research Institute, Barts & the London School of Medicine and Dentistry, Queen Mary University of London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Federico Roncaroli 4 Goeffrey Jefferson Brain Research Centre, Division of Neuroscience, Faculty of Biology, Medicine and Health, University of Manchester , Manchester, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Shannon W Davis 5 Department of Biological Sciences, University of South Carolina , Columbia, SC, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Carles Gaston-Massuet 1 Centre for Endocrinology, William Harvey Research Institute, Barts & the London School of Medicine and Dentistry, Queen Mary University of London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Carles Gaston-Massuet For correspondence: c.gaston-massuet{at}qmul.ac.uk Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Adamantinomatous craniopharyngiomas (aCPs) are complex intracranial neoplasms that generally arise in the sellar and suprasellar region of the brain which affect the endocrine and nervous systems causing severe sequelae. Activating mutations resulting in degradation-resistant forms of β-catenin ( CTNNB1 ) have been shown to be the main driver for many of these neoplasms. However, the underlying genetic driver for a proportion of these tumours is still unknown. Using murine transgenic models, we show that genetic disruption of the Wnt-antagonist and tumour suppressor, adenomatous polyposis coli ( Apc ), within the pituitary progenitors/stem cells, leads to pituitary tumours that closely resemble human aCPs. These tumours present classic histopathological hallmarks of aCPs, such as large deposits of wet keratin, stellar-reticular-like cells, large cystic components and clusters of accumulating nucleo-cytoplasmic β-catenin that are slow-dividing and exhibit a secretory phenotype. We show that a hypomorphic allele of Apc is sufficient for tumour development, indicating that disruption of Apc function can lead to aCP formation. Moreover, we identify that bi-allelic loss of Apc in the Sox2+ve pituitary stem cells is sufficient to initiate tumour formation, indicating that Sox2+ve stem cells are the cell origin of these Apc -driven aCPs. Transcriptomic analyses of early tumour-initiating cells revealed that Apc- driven clusters of accumulating β-catenin undergo senescence-associated secretory phenotype (SASP), which is p21-mediated and results in secretion of inflammasome, angiosome and developmental growth factors. Our data unequivocally show a causal role for the disruption of the tumour suppressor Apc as a primary driver of aCPs independent of mutations in β-catenin. We provide murine models representing a novel genetic subtype of human aCPs offering insights into aCP pathogenesis. Our work reinforces the importance of genetic testing for mutations in APC in patients with aCPs and identifies a potential need to screen patients with familial adenomatous polyposis (FAP) or spectrum of APC -pathogenic syndromes for aCPs in early life. Introduction Craniopharyngiomas (CPs) are complex neoplasms classified by the World Health Organisation (WHO) as grade 1 benign epithelial tumours typically located within the sellar or suprasellar region of the brain 1 . They are classified into two distinct subtypes: papillary craniopharyngioma (pCPs) which account for around 10% and adamantinomatous craniopharyngioma (aCPs) representing the remaining 90% of all CPs 2 . Molecularly, 90% of pCPs are driven by mutations in BRAF p.V600E 3 , 4 , whilst the majority of aCPs are driven by mutations in the CTNNB1 gene encoding for β-catenin. Although rare, with an incidence of 1.7 per 1,000,000 2 , 5 , aCPs are the most common suprasellar and non-neuroepithelial intracranial tumour in childhood and have been shown to originate from progenitors/stem cells of the pituitary gland 6 , 7 . aCPs can occur at any age 8 but have a bimodal age of presentation between 5-14 and 55-70 years old 9 . Although classified as benign, aCPs often exhibit infiltrative behaviour, invading critical neighbouring structures such as the hypothalamus and optic and cranial nerves. This makes total surgical resection technically difficult resulting in a high rate of recurrence 2 , 9 . Current treatment of aCPs is limited to surgery combined with radiotherapy, both of which are associated with high morbidity and significant disabling side effects. The prevalence of mutations in β-catenin in human aCPs varies from 30% to 100% depending on the study 10 - 16 . The variability in mutation frequency may be due to small patient cohorts and/or the use of sequencing methods lacking the high depth of reading required for the detection of low allelic frequency variants in these highly heterogeneous tumours. Consequently, main genetic drives for a proportion of aCPs remains unknown. Previous transgenic murine models have been instrumental to understanding aCP pathogenesis 6 , 7 , 17 , 18 . Pituitary tissue-specific expression of a degradation-resistant form of β-catenin in murine pituitary stem cells has demonstrated that mutations in β-catenin causally drive development of aCPs 6 . These tumours have similar histopathological and molecular features to human aCPs, including the presence of whorl-like clusters or clusters of accumulating nucleo-cytoplasmic β-catenin that overactivate the canonical Wnt/β-catenin pathway. These clusters have been shown both in mice 18 and human 19 , 20 to undergo oncogene-mediated senescence and act as secretory hubs to modify the tumour microenvironment 18 , 19 . β-catenin is the central component of the Wnt/β-catenin pathway, which is crucial for development and stem cell homeostasis, with its dysregulation leading to tumour formation 21 - 23 . In the absence of secreted Wnt ligands, β-catenin is phosphorylated by a destruction complex formed by the scaffold protein Axin2, adenomatous polyposis coli (APC) and the kinase, GSK-3β. At this stage, β-catenin is phosphorylated by GSK-3β, ubiquitinated and targeted for proteasomal degradation, thereby regulating the levels of active β-catenin present in the cell 24 . Mutations in negative regulators of the Wnt pathway, that function to degrade β-catenin, such as adenomatous polyposis coli ( APC ), result in increased Wnt/β-catenin signalling, and are involved in the pathogeneisis of numerous cancers 25 . Indeed, both somatic and germline mutations in APC are the main drivers of colon carcinoma. Germline mutations cause familial adenomatous polyposis (FAP) 26 and the related spectrum of APC -pathogenic syndromes such as Gardner 27 and Turcot Syndrome 28 which are characterised by colonic adenomas alongside tumours within the brain, bone and skin 29 , 30 . Recently, an association between FAP and aCPs has been reported in 2 separate studies 31 , 32 . These reports, using whole exome sequencing, identified 3 cases of aCPs harbouring germline mutations in APC , implicating this negative regulator of Wnt/β-catenin pathway in the pathogenesis of aCPs. A further possible association between FAP and aCPs has been hypothesised as some patients with Gardner syndrome, which carry germline mutations in APC , have been reported to develop ectopic aCPs 33 - 39 . However, a causal role of APC mutations in the formation of these aCPs, could not be determined due to the lack of tumour genomic sequencing data for these patients 39 - 42 . Moreover, a recent study that analysed two transgenic models to genetically delete Apc concluded that genetic deletion of Apc in rodent pituitary progenitors was not tumorigenic resulting in the absence of aCP formation even in older mice 18 . Therefore, although an association between aCPs and mutations in APC exists, the causal role for the dysfunction of Apc in aCP pathogenesis has remained elusive so far. To demonstrate the impact of Apc disruption in aCP pathogenesis, we used murine transgenic models combined with transcriptomic analyses of early aCP tumour-initiating cells to demonstrate a causal role of Apc disruption in aCP pathogenesis. We show that biallelic loss of the truncated form of Apc within pituitary progenitors, by deleting Apc exon 15 , results in severe intrauterine aCPs that are not compatible with life. Generation of an Apc hypomorphic allele results in aCPs postnatally that accurately recapitulate aCP disease progression, presenting with key histological and molecular hallmarks of human aCPs, as well as clinically relevant tumour-induced hypothalamic obesity. Transcriptomic analyses of early tumour-initiating cells revealed that the disruption of Apc within pituitary stem cells leads to clusters of accumulating nucleo-cytoplasmic β-catenin that undergo a senescence associated secretory phenotype (SASP). We prove that this secretory signature consists of specific pro-inflammatory mediators, chemokines, cytokines and developmental growth factors. Importantly, these Apc- driven tumour-initiating cells exhibit transcriptional signatures that could be pharmacologically targeted. Our work demonstrates a direct causal effect of Apc disruption as a main driver of aCPs independent of β-catenin mutations. We provide further understanding of the underlying genetics of aCPs and present murine models representing a novel genetic subtype of human aCPs. Our results reinforce the need for genetic testing for mutations in APC in patients with aCP and the importance of increased surveillance for patients with FAP or APC -related syndromes for the development of extracolonic tumours, such as aCPs. Results Bi-allelic loss of Apc exon 15 in the pituitary embryonic precursors leads to severe aCPs during embryogenesis To determine the causal role of APC in craniopharyngioma formation, we employed a murine transgenic approach to delete exon 15 of Apc specifically in embryonic pituitary progenitor cells. We used the conditional Apc allele in which exon 15 has been flanked with LoxP sites Apc LoxP(Exon15)/LoxP(Exon15) 43 to generate a conditional truncated form of Apc . Exon 15 of Apc encompasses 2200 amino-acids, including the functional domain responsible for β-catenin degradation and the C-terminal domain involved in microtubule binding 43 . To conditionally delete Apc within embryonic pituitary progenitor cells, we used the Prop1:Cre driver 44 , 45 , that expresses Cre within pituitary progenitors from embryonic day (e) 10 of gestation, to generate homozygotes Prop1:Cre;Apc LoxP(Exon15)/LoxP(Exon15) (hereafter referred to as Cre:Apc LoxP15/15 for simplicity). Homozygotes Cre:Apc LoxP15/15 pups died perinatally, with none of the mutant pups reaching postnatal day 7. Dissections of pituitary glands revealed large tumours in all Cre:Apc LoxP15/15 embryos compared to their wild type (Wt, Apc +/+ ) littermates ( Fig. 1a ). Histological analyses using haematoxylin and eosin staining (H&E) and antibodies against terminally differentiated hormone producing cells, revealed that Cre:Apc LoxP15/15 mutant embryos exhibit large pituitary tumours that are almost devoid of all terminally differentiated hormone producing cells ( Fig. 1b ). These tumours are characterised by aCP-histopathological features such as wet keratin and whorl-like structures consisting of cell clusters of nucleo-cytoplasmic positive β-catenin ( Fig. 1d ). Abnormal larger pituitary glands were observed as early as embryonic day (e) 11 and progressively worsen throughout gestation, exhibiting statistically significant increase in pituitary volume ( Fig. 1c ) secondary to increased mitotic index (MI) ( Fig. 1 e-f ). To further characterise these tumours, we performed double immunofluorescence with β-catenin and the proliferation marker phospho-histone H3 (α-pHH3) or the pituitary stem cell markers Sox2 or Sox9, as aCP-histological hallmarks 6 , 17 , 46 , 47 . Double immunofluorescence at e13.5 and e18.5 revealed large clusters of nucleo-cytoplasmic accumulating β-catenin, which were negative for pHH3. Notably most of the dividing cells were located surrounding the β-catenin+ve cell clusters ( Fig. 2 a-b ). As previously described in β-catenin-driven aCP tumours 6 , 7 , these β-catenin+ve clusters express the pan-pituitary stem cell marker Sox2 ( Fig. 2 c-d ), but are negative for the pituitary transcription factor Sox9. Instead, Sox9+ve cells were localised around the β-catenin+ve cell clusters ( Fig. 2 e-f ). We confirmed the efficiency of recombination of the exon 15 of Apc by RT-qPCR and identified 100% deletion of exon 15 by our Prop1:Cre driver line ( Supplementary Fig. 1d ). Together, our results demonstrate that bi-allelic loss of Apc exon 15 within pituitary progenitor cells, results in intrauterine aCPs associated with early postnatal lethality due to tumour size and severity, with absence of differentiation of hormone secreting cells in the anterior gland. Download figure Open in new tab Fig 1. Genetic deletion of Apc exon15 in the pituitary progenitors results in large embryonic non-secreting pituitary tumours with classic histological hallmarks of aCPs. a ) Wholemount picture of dissected pituitaries at embryonic day e18.5 of a wild type (Wt, Apc +/+ ) compared to mutant Cre;Apc LoxP15/15 exhibiting a large tumour. b ) Coronal sections through the pituitary gland at e18.5 stained with H&E reveal a large hyperplastic tumour with epithelial whorl-like structures (arrows) within the tumour parenchyma in the Cre;Apc LoxP15/15 (right column) compared to Wt pituitary glands (left column). IHC against GH, POMC (melanotrophs and ACTH), PRL, TSH and LH reveals that the Cre;Apc LoxP15/15 tumours are largely devoid of hormone-producing cells, apart from some scattered positive foci of POMC and TSH (arrowheads). c ) Volumetric quantifications indicate that the Cre;Apc LoxP15/15 mutant pituitaries have statistically significant larger volumes compared to Wt littermates at both e15.5 and e18.5 of gestation. d ) Immunofluorescence against β-catenin reveals clusters of nucleo-cytoplasmic β-catenin accumulating cells which are hallmarks of aCPs in the developing pituitary gland at e13.5, e15.5 and e18.5 in the mutant Cre;Apc LoxP15/15 (white arrowheads) compared to the membranous only β-catenin staining in the Wt pituitaries. e ) IHC against the proliferation maker, phospho-histone H3 (α-pHH3), reveals increase in pHH3-postive cells in the Cre;Apc LoxP15/15 mutant pituitaries at e13.5, e15.5 and e18.5 compared to Wt. Quantification of the percentage of pHH3+ve cells revealed a statistically significant increased mitotic index (MI) in Cre;Apc LoxP15/15 compared to their Wt littermates. Right-hand column images in d & e are enlarged images of the squared boxes of the images in the central column. P values in c and f were obtained using unpaired two-tailed Student’s T -test and data represented as mean ± SEM, from 3/4 pituitaries per genotype. Images are representative of 4-5 embryos per genotype. Abbreviations: AL, anterior lobe; LH, luteinizing hormone; GH, growth hormone; H&E, haematoxylin and eosin; IHC, immuno-histochemistry; MZ, marginal zone; PL, posterior lobe; POMC, Pro-opiomelanocortin; PRL, prolactin; SB, sphenoid bone; TSH, thyroid-stimulating hormone. Scale bar in b represents 50 μm; and in d represenst 200 μm. Download figure Open in new tab Fig 2. Genetic deletion of Apc exon15 ( Cre;Apc LoxP15/15 ) leads to aCP histological hallmarks such as cell clusters of accumulating nucleo-cytoplasmic β-catenin. These express the stem cell marker Sox2, are negative for the Sox9 and have low proliferative capacity. a-f ) Histological sections through the pituitary gland at e13.5 ( a, c, e ) and e18.5 ( b, d, f ) immune-stained with α-pHH3 ( a, b ), α-Sox2 ( c, d ) and α-Sox9 ( e, f ). a, b ) Double immunofluorescence against pHH3 (red) and β-catenin (green) reveals the presence of clusters of accumulating nucleo-cytoplasmic β-catenin (green) which do not express the proliferation marker pHH3 in the Cre;Apc LoxP15/15 . Proliferating cells (pHH3+ve cells, white arrowheads) were always found outside of the β-catenin cell clusters at e13.5 ( a ) and e18.5 ( b ). c, d ) Double immunofluorescence against β-catenin (green) and α-Sox2 (red) reveals that clusters of accumulating β-catenin express the pituitary stem cell marker Sox2 (white arrowheads). e, f ) Double immunofluorescence against β-catenin (green) and α-Sox9 (red) shows that clusters of accumulating β-catenin do not express transcription factor Sox9 but rather, the Sox9+ve cells are found surrounding the β-catenin+ve clusters. Note that no clusters of β-catenin were observed in any of the Wt pituitaries ( Apc +/+ ) with β-catenin being localised to the cell membrane. Magnified images in the right columns in a to f represent the white-dashed squared areas in the middle column panels. Images are representative of 4 embryos per genotype per each gestational stage. Abbreviations: AL, anterior lobe; MZ, marginal zone; PL, posterior lobe. Scale bars in a, c, e represent 200 μm and in b, d, f represent 100 μm. A hypomorphic allele for Apc results in postnatal aCP tumour formation To overcome the early lethality observed in the Cre:Apc LoxP15/15 animals, we took a genetic approach to generate another mutant allele of Apc . To achieve this, we utilised the Apx LoxP(Exon1-15) line in which LoxP -sites have been introduced to flank all the Apc exons, from exon 1 to exon 15, enabling deletion of the entire Apc locus 48 . Homozygotes Prop1:Cre : Apc LoxPExon 1-15/LoxPExon1-15 (hereby referred to as Cre:Apc LoxP1-15/1-15 for simplicity) exhibited enlarged pituitary glands due to increased MI ( Supplementary Fig. 1 a-b ) with decreased number of growth hormone-producing cells ( Supplementary Fig. 1 a ). However, no tumour formation occurred in any of the mutant mice ( Cre:Apc LoxP1-15/1-15 ) analysed after 2 years, other than hyperplasia of the anterior pituitary gland (data not shown). This prompted us to investigate the efficiency of Cre recombination of exons 1-15 at the Apc locus by RT-qPCR in our homozygote Cre:Apc LoxP1-15/1-15 pituitaries. Importantly, we identified that 16.37% of Wt Apc exon 15 mRNA was being produced, indicating inefficient recombination of the Apc locus ( Supplementary Fig. 1d ). This result indicates that a small amount of Wt Apc mRNA is sufficient to prevent tumour formation, resulting in pituitary hyperplasia instead of tumours. Hence, we reasoned that further reduction of Wt Apc levels by combining the Cre:Apc LoxP15/+ (with 100% recombination efficiency) with the Cre:Apc LoxP 1-15/+ alleles to generate a compound heterozygote Cre ; Apc LoxP1-15/15 could result in a milder phenotype compared to the Cre:Apc LoxP15/15 . Indeed, all Cre:Apc LoxP1-15/15 compound mutants survived to adult compared to the early perinatal lethality observed in the Cre:Apc LoxP15/15 pups ( Fig. 1 ). All Cre:Apc LoxP1-15/15 compound mutants developed large pituitary tumours postnatally, leading to tumour-associated co-morbidities such as severe obesity and reduced life expectancy ( Fig. 3 a-f ). Obesity secondary to tumour formation was apparent in all mutant Cre:Apc LoxP1-15/15 mice from 4 months of age( Fig. 3 c-d ), an important phenotype that has not being observed in previously published murine aCP models 6 . Histological analyses of these tumours revealed the presence of large cystic components, stellar reticular-like cells and nucleo-cytoplasmic β-catenin+ve cell clusters, all of which are histological diagnostic hallmarks of aCPs ( Fig. 3b ). Interestingly, Kaplan Meier survival curves revealed a gender difference in survival, with female mice surviving longer than males ( Fig. 3 e-f ). Download figure Open in new tab Fig 3. A hypomorphic allele of Apc ( Cre:Apc LoxP1-15/15 ) develops large pituitary tumours postnatally. a ) Picture after removal of the scalp and brain of 6 month old Cre:Apc LoxP1-15/15 mutants revealed pituitary tumours (white arrows). b ) H&E sections of the tumours reveal large cystic components (Cyst) and some cells reminiscent of stellate reticulum (SR) cells. IHC against β-catenin shows cells with nuclear positivity within some cell clusters (black arrows). c-d ) Male and female hypomorphic animals exhibit statistically significant weight gain starting from 4 months of age, with all mutant animals exhibiting obesity compared to their Wt littermates ( d ). Kaplan Meier survival curves from males ( e ) and females ( f ) show that hypomorphic Cre:Apc LoxP1-15/15 mutant animals have shorter lifespans compared to their Wt littermates. Note that males ( e ) have a shorter life span compared to females ( f ), with a 50% probability of survival at 6 months (180 days), whilst females exhibited a longer life span (320 days). P values in d represent ***<0.001; ** <0.01 and were obtained using unpaired two-tailed Student’s T -test and data represented as mean ± SEM. e & f P values of survival curves were obtained by a long-rank Mantel-Cox test. Abbreviations: H&E, haematoxylin and eosin; SR, stellate reticulum cells. Scale bars in b represent 100 μm. Characterisation of tumours at 3 months of age also revealed all the typical histological diagnostic hallmarks of human aCPs including cysts, nucleo-cytoplasmic β-catenin+ve cell clusters, large foci of wet keratin and increased MI ( Fig. 4 a-e ). Immunostaining of the pituitary gland for terminally differentiated hormones, revealed the absence of hormone-producing cells within the developing tumour in keeping with the non-hormone secreting nature of aCPs ( Fig. 4d ). H&E staining revealed wet keratin deposits, cysts and nucleo-cytoplasmic β-catenin+ve cell clusters often surrounding the wet keratin foci ( Fig. 4e ). To analyse the early tumour characteristics of the Cre:Apc LoxP1-15/ 15 hypomorphs at e18.5, double immunofluorescence against β-catenin and pHH3, Sox2 or Sox9 was performed. These early tumours exhibit clusters of accumulating nucleo-cytoplasmic β-catenin, which are negative for pHH3, with dividing cells localised to the surrounding of β-catenin+ve cell clusters ( Fig. 5a ). In contrast with the perinatal lethal Cre:Apc LoxP15/15 model, the β-catenin+ve clusters of the Cre:Apc LoxP1-15/15 tumours were smaller ( Supplementary Fig. 2 ). The β-catenin+ve cell clusters were all positive for the pituitary stem cell marker Sox2, and negative for Sox9, with Sox9+ve cells localised around the β-catenin+ve cell clusters ( Fig. 5a ). Clonogenic assays found a statistically significant increase in the number of stem cell colonies and the number of cells per colony in mutant Cre:Apc LoxP1-15/15 derived pituitary stem cells at e18.5 compared to Wt pituitaries, indicating that mutant derived pituitary stem cells have more proliferative capacity ( Fig. 5b ). At this embryonic stage, IHC for terminally differentiated hormones revealed a decrease in hormone-producing cells compared to Wt with a clear decrease in GH, TSH, FSH and LH, whilst no differences were observed in other hormones ( Fig. 5c ). Next, RT-qPCR was used to identify the level of recombination in the Cre:Apc LoxP1-15/15 compound mutants. This identified that Wt Apc exon15 mRNA was present in small amounts (7.5% compared to Wt cells) whilst the exon 15 from the Cre:Apc LoxP15/15 was fully deleted ( Supplementary Fig. 1d ). This result indicates that the Cre:Apc loxP1-15/15 expresses small amounts of Wt Apc and is therefore the hypomorphic allele of Apc. These low levels of Wt Apc are insufficient to prevent tumour formation in compound Cre:Apc LoxP1-15/15 animals, but result in a milder phenotype compared to Cre:Apc 15/15 embryonic aCPs which exhibits 100% recombination of the Apc-exon15 and are not viable postnatally. Download figure Open in new tab Fig 4. Mid-stage tumours from Cre:Apc LoxP1-15/15 hypomorphs exhibit classic histological hallmarks of aCPs. a ) Picture after removal of the scalp and brain in 3 month old, Cre:Apc LoxP1-15/15 mutants revealed hyperplastic pituitaries with small tumours (white arrows & T). b ) H&E staining of coronal sections through the Cre:Apc LoxP1- 15/15 mutant pituitaries revealed developing tumours within the pituitary parenchyma of differing sizes (marked as T). These tumours have increased pHH3+ve cells, resulting in a higher mitotic index (MI) ( c ). d ) IHC against GH, POMC (melanotrophs and ACTH), PRL, TSH and FSH reveals that the developing tumours are devoid of hormone-producing cells. e ) H&E staining of coronal sections through Cre:Apc LoxP1-15/15 mutant pituitaries revealed histological hallmarks of aCPs, such as deposits of wet keratin (Wk) and small cysts (Cy). IHC against β-catenin in mutant pituitaries revealed the presence of cell clusters of nucleo-cytoplasmic β-catenin (black arrowheads) surrounding nodules of wet keratin (Wk, black arrows). P value in ( c ) was obtained using unpaired two-tailed Student’s T -test and data represented as mean ± SEM. Images are representative of 5 mutant pituitaries per genotype. Abbreviations: AL, anterior lobe; FSH, follicle stimulating hormone; GH, growth hormone; PL, posterior lobe; PRL, prolactin; POMC, pro-opiomelanocortin; MZ, marginal zone; RAL, right anterior lobe; LAL, left anterior lobe; T, tumours; TSH, thyroid-stimulating hormone; Wk, wet keratin. Scale bars in b & d represent 100 μm, and in e represents 50 μm. Download figure Open in new tab Fig 5. Embryonic pituitaries from Cre:Apc LoxP 1-15/15 embryos exhibit all the features of early aCP development. a ) Double immunofluorescence of coronal sections through the developing pituitary gland from the Cre:Apc LoxP 1-15/15 embryos at e18.5 against pHH3, Sox2 and Sox9 with β-catenin. Clusters of nucleo-cytoplasmic β-catenin +ve cells are present throughout the pituitary parenchyma (green, white arrows) with pHH3+ve cells (red, white arrowheads) scattered around the clusters of β-catenin (white arrows in the magnified image). All clusters of accumulating β-catenin express the pituitary stem cell marker Sox2 (red, white arrowheads) and the Sox9+ve cells are located around the β-catenin+ve clusters. The right column images are magnified images from the white squared areas in the middle column. b ) Pituitary stem cell culture from hypomorphic Cre:Apc LoxP 1-15/15 mutants exhibit higher clonogenic potential evidenced by both increased number of colonies and number of cells per colony compared to Wt pituitary stem cells. c ) IHC against β-catenin in coronal sections through the pituitary and brain at e18.5 revealed clusters of cells of accumulating β-catenin in Cre:Apc LoxP 1- 15/15 (right column, black arrowheads) compared to Wt (left column). IHC against GH, TSH, LH and FSH reveals that Cre:Apc LoxP 1-15/15 hypomorphic mutant pituitaries exhibit a reduction in these hormone-producing cells compared to their Wt littermates, whilst POMC (melanotrophs and ACTH) and PRL show similar expression between genotypes. d ) In situ hybridisation on coronal sections through the pituitary gland of Cre:Apc LoxP1-15/15 mutants reveals upregulation of the canonical downstream Wnt-effector Axin2 and the senescence marker p21 in a cell cluster fashion. The developmental factors Shh, Fgf8, Fgf3 and Wnt6, Wnt7a were highly upregulated within cell clusters throughout the pituitary parenchyma. Right column panels are magnified images from the dotted squared areas in the left column. P values in b were obtained using unpaired two-tailed Student’s T -test and data represented as mean ± SEM. Images are representative of 5 embryos per genotype. Abbreviations: AL, anterior lobe; LH, luteinising hormone; GH, growth hormone; IHC, immuno-histochemistry; MZ, marginal zone; PL, posterior lobe; PRL, prolactin; POMC, Pro-opiomelanocortin; SB, sphenoid bone; TSH, thyroid-stimulating hormone. Scale bars in b represent 50 μm; and in c and d represent 100 μm. β-catenin-mutated+ve aCPs are characterised by the presence of clusters of accumulating nucleo-cytoplasmic β-catenin that undergo senescence secretory associated phenotype (SASP) and secrete factors into the tumour microenvironment 18 , 19 , 49 . We, therefore, analysed by in situ hybridisation the expression of some markers of β-catenin+ve clusters previously reported in mouse and human aCPs 6 , 17 . All clusters analysed from the Cre:Apc loxP1-15/15 hypomorphic pituitaries expressed the canonical Wnt-activating target, Axin2 , indicating β-catenin+ve cell clusters over-activate the Wnt canonical pathway. Moreover, these clusters over-express the developmentally secreted factors Wnt6, Wnt7a, Fgf3, Fgf8 and Shh in a cluster fashion ( Fig. 5d ). Importantly, these clusters upregulate the senescence associated marker Cdkn1 ( p21 ) in accordance with activation of SASP described in both human 19 , 49 and murine aCPs 17 , 18 . Taken together, our results demonstrate that a hypomorphic allele of Apc leads to tumour formation postnatally, exhibiting all the characteristic histological and molecular features of aCP tumours, including associated comorbidities such as tumour-induced obesity. These results further demonstrate a causal role of Apc dysfunction in the development of aCPs. Biallelic loss of Apc exon 15 in the Sox2+ve pituitary stem cells is sufficient to initiate aCP tumour formation Previous studies using a conditional transgenic line have shown that Sox2+ve pituitary stem cells bearing oncogenic β-catenin ( Ctnnb1 LoxpExon3/ + ) can initiate aCP tumour formation 7 . To further prove a causative role of Apc in aCP formation, we employed an inducible Sox2 CreERT2/+ transgenic line 50 in which tamoxifen (Tam) induced Cre is achieved within Sox2+ve stem cells. To enable the deletion of exon 15 of Apc we crossed the Sox2 CreERT2 /+ to the Apc LoxP(Exon15)/LoxP(Exon15) strain to generate Sox2 CreERT2/+ ;Apc LoxP(Exon15)/LoxP(Exon15) embryos (hereafter refer to as Sox2 CreERT2/+ ;Apc LoxP15/15 for simplicity). Deletion of Apc exon 15 was achieved with 3 consecutive daily injections of Tam at 0.1 mg/gr/mouse during pregnancy starting at e12.5, and embryos were analysed at e18.5 ( Fig. 6a ). Download figure Open in new tab Fig 6. Deletion of Apc exon15 in the Sox2+ve pituitary stem cells results in early aCP formation. a ) Experimental diagram: to achieve deletion of Apc exon15 ( Sox2 CreERT2/+ ;Apc LoxP15/15 ), pregnant females were Tam injected (0.1 mg/g of body weight) with 3 consecutive daily injections at days e12, e13 and e14 of pregnancy. Embryos were dissected at e18.5 and pituitary glands were analysed by in situ hybridisation and immunostaining. b ) IHC against β-catenin revealed the presence of multiple cell clusters of nucleo-cytoplasmic accumulating β-catenin (arrowheads). In situ hybridisation on consecutive sections for Axin2 and the senescence marker p21 reveals the upregulation of Axin2 and p21 within the β-catenin positive clusters (arrows). Pictures in the right column are magnified images from the squared dashed boxes in the left column. c ) Double immunofluorescence against Sox2, Sox9, pHH3 (red) and β-catenin (green) revealed that Tam-induced Sox2 CreERT2/+ ;Apc LoxP15/15 embryos develop early aCP lesions characterised by clusters of nucleo-cytoplasmic accumulating β-catenin cells (green, white arrowhead) which co-express the pituitary stem cell marker Sox2. Sox9+ve cells and proliferating cells (pHH3+ve cells) are found around the β-catenin+ve cell clusters (white arrows). Images are representative of 3 embryos per genotype. Abbreviations: AL, anterior lobe; IL, intermediate lobe; IHC, immuno-histochemistry; MZ, marginal zone; PL, posterior lobe; Tam, tamoxifen. Scale bars in b and c represent 50 μm. Interestingly, Tam-induced Sox2 CreERT2/+ ;Apc LoxP15/15 embryos exhibited hyperplasia of the anterior pituitary gland with the presence of clusters of accumulating nucleo-cytoplasmic β-catenin ( Fig. 6 b-c ). In situ hybridisation on consecutive histological sections revealed β-catenin+ve clusters expressing Axin2 and the senescence marker p21 ( Fig. 6b ) similar to previously discussed Apc -driven models ( Fig. 2 & Fig.5 ). These clusters are Sox2+ve, have low proliferating capacity (negative for pHH3) and are surrounded by Sox9+ve cells ( Fig. 6c ) in keeping with aCPs 6 , 7 , 19 . These results indicate that the Sox2+ve pituitary stem cells bearing bi-allelic loss of the tumour suppressor Apc ( Sox2 CreERT2/+ ;Apc LoxP15/15 ) are sufficient to initiate aCP tumour formation resulting in clusters of accumulating β-catenin that express diagnostic hallmarks of aCPs. Apc -driven cell clusters with nuclear β-catenin translocation undergo SASP and secrete inflammatory mediators, chemokines, cytokines, keratins and developmental growth factors β-catenin-mutated+ve aCPs are characterised by whorl-like clusters of accumulating nucleo-cytoplasmic β-catenin which undergo SASP 7 , 18 and act as hubs of secreting factors into the tumour microenvironment 7 , 18 , 19 . To investigate whether Apc -driven tumours contain clusters that act as signalling hubs, and to compare their molecular signatures to human β-catenin-mutated+ve aCPs, we performed transcriptomic analyses from isolated cell clusters from our Cre:Apc LoxP15/15 model at embryonic stages aiming to identify early tumour transcriptional signatures. To isolate β-catenin+ve cell clusters we utilised the transgenic line Tcf/Lef-1:H2B-GFP as a readout of activated Wnt-pathway 51 . The Tcf/Lef-1:H2B-GFP transgenic line (hereafter referred to as Lef-1:GFP for simplicity) harbours a knock-in of the Wnt/β-catenin signalling reporter construct containing 6 Tcf/Lef1 response elements that drive the expression of GFP enabling fluorescent marking of Wnt-responsive cells. This transgenic line has been shown to accurately recapitulate the known pattern of Wnt-responsive cells and, therefore, represents an accurate read-out of activated Wnt/β-catenin canonical pathway 51 . We crossed our model Cre:Apc LoxP15/15 to the Lef-1:GFP reporter strain to generate Cre:Apc LoxP15/15 ;Lef-1:GFP compound mutants and assessed if the β-catenin+ve clusters expressed GFP ( Fig. 7 a-b ). Download figure Open in new tab Fig 7. Transcriptomic analyses of early Apc -driven β-catenin+ve GFP+ve cell clusters show transcriptional signatures involved in inflammation, developmental growth factors and SASP. a ) Diagram representing experimental design: mRNA sequencing from β-catenin+ve clusters from Apc -driven tumours ( Cre:Apc LoxP15/15 ;Lef-1:GFP ) was performed from the FAC-sorted GFP+ve fraction cells (β-catenin+ve GFP+ve cell clusters) and compared to Sox2+ve stem cells from Tam-induced Sox2 CreERT2/+ ;Rosa26 TdTomato (Tomato+ve fraction cells). b ) Double immunofluorescence against β-catenin (green) and α-GFP (red) revealed that all the nucleo-cytoplasmic β-catenin+ve cell clusters (white arrows) express GFP under the Lef1 reporter sequence of the Cre:Apc LoxP15/15 ;Lef-1:GFP . c ) Hierarchical clustering of the top 5000 most variable genes separates the cell population’s transcriptome from the Apc -driven β-catenin+ve GFP+ve cell clusters from control Sox2 CreERT2/+ ;Rosa26 TdTomato/+ . d ) Volcano plot showing differential gene expression between the β-catenin+ve GFP+ve cell clusters and control Sox2+ve pituitary stem cells. Highlighted in red are key upregulated genes such as the negative regulators of Wnt/β-catenin pathway Notum, Dkk1; the downstream effectors of activated Wnt/β-catenin pathway such as Axin2, Sp5, Wnt7a indicating overactivation of the canonical Wnt/β-catenin pathways; the developmental secreted factors Fgf3, Fgf4, Fgf15 and Bmp4 are found upregulated in addition to the senescence-associated factor p21 (Cdkn1a) . In line with aCPs being non-secreting tumours, pituitary terminal differentiation factors such as Gh, Pomc, Lhb, Tsh and Prl were downregulated. Abbreviations: Gh , growth hormone; FACs, fluorescent activating cell sorting; Lhb , luteinising hormone; Prl, prolactin; Tsh, thyroid-stimulating hormone. Scale bar in b represents 40 μm and the images are representative of 4/5 mutant embryos. Double immunofluorescence against β-catenin and GFP revealed that all β-catenin+ve clusters, which over-activate Wnt-downstream targets, expressed GFP ( Fig. 7b ). Moreover, no GFP+ve cells were observed outside of these cell clusters, enabling accurate and specific fluorescent labelling of the β-catenin+ve cell clusters using GFP and the ability to perform purification using fluorescence activated cell sorting (FACs) ( Fig. 7a ). Tumours were dissociated to single cells and the GFP+ve fraction (β-catenin+ve GFP+ve cell clusters) was FAC-sorted before mRNA was extracted, purified and submitted for transcriptomic analyses. The mRNA from the GFP+ve cell fraction was compared to mRNA isolated from Wt pituitary stem cells from the Sox2 CreERT2/+ ;Rosa26 LoxPSTOPLoxPTdTmT which allows for FAC-sorting of Sox2+ve cells expressing the red fluorescent protein TdTomato (or TdTmt for simplicity) ( Fig. 7a ). Hierarchical clustering of the most variable expressed genes distinguished transcriptional differences between the β-catenin+ve-GFP+ve cluster cells from that of the Sox2+ve TdTmt+ve cell population ( Fig. 7c ). Volcano plot analysis of differentially expressed genes revealed that Apc -driven clusters (β-catenin+ve GFP+ve) downregulate pituitary hormones such Gh, Tsh, Pomc, Prl and Lh ( Fig. 7d ) in accordance with the non-secreting nature of aCPs and is in keeping with our previous immunostaining analyses ( Fig. 1b & Fig. 4d ). Upregulated genes indicated that β-catenin+ve GFP+ve cell clusters activate the Wnt/β-catenin canonical pathway: with increase in the Wnt-downstream targets Axin2, Lef1, Sp5, Tcf4 ; and the secreted Wnt ligands: Wnt7a, Wnt6, Wnt10a, Wnt9b, Wnt11, Wnt16 and their receptors and co-receptors Fzd4, Fzd10, Lgr6 . Moreover, the secreted Wnt antagonists Notum, Dkk1, Dkk2, Dkk4, Dkk3 and Wif1 were found to be highly upregulated in the Apc- driven β-catenin+ve GFP+ve cell clusters ( Supplementary Fig. 3a ). We identified that genes such as the secreted fibroblast growth factor ( Fgfs ): Fgf3, Fgf15, Fgf4, Fgf8, Fgf20 and Fgf17 , the bone morphogenic factors: Bmp4, Bmp8a, Bmp2, Bmp5, Bmp15 and Shh were found highly upregulated ( Supplementary Fig. 3b-c ). Keratins and keratin-associated proteins were highly upregulated by the Apc -driven β-catenin+ve GFP+ve clusters, which may contribute to the large deposits of wet keratin observed in our Apc -mutant model ( Fig. 4 e , Supplementary Fig. 4 a-b ). We identified that the GFP+ve β-catenin+ve clusters secrete a large number of interleukins ( Il6,Ilb, Il2b, Il36b , Il10, Il16, Il15 ) and chemokines ( Ccl2, Ccl3, Ccl4, Ccl5, Ccl7, Ccl11, Cxcl9, Cxcl15, Cxcl3, Cxcl1, Cxcl11 ) which have been reported to have proinflammatory, chemotactic, immunosuppressor and pro-angiogenic functions during tumour development and are the main components of SASP 52 - 55 ( Supplementary Fig. 5 a-c ). In agreement with SASP, we identified that the SASP-associated factor p21 ( Cdkn1a ) was highly upregulated in the Apc -driven β-catenin+ve GFP+ve cell clusters which was also confirmed by in situ hybridisation ( Fig. 5d ). Some of the above-upregulated genes were found overexpressed in our Apc -model by in situ hybridisation in a cell cluster fashion ( Fig. 5d ) and mRNA-seq data was validated by RT-qPCR using mRNA isolated from Apc- driven GFP+ve β-catenin+ve cell clusters ( Supplementary Fig. 4c ). Unsupervised gene set enrichment analyses (GSEA) identified pathways upregulated by the Apc -driven β-catenin+ve GFP+ve cell clusters, such as positive regulation of inflammation, enrichment of IL2-Stat5 and IL6-Jak-Stat3 signalling pathways. These pathways are important in immunomodulation, inflammation and angiogenesis 53 - 55 ( Fig. 8a ), reinforcing the hypothesis that β-catenin+ve GFP+ve clusters act as paracrine secreting hubs. Genes involved in positive regulation of TNF-alpha as well as KRAS and p53 pathways were found upregulated in cluster cells ( Fig. 8a ). We then compared our mRNA-seq transcriptomic data from our Apc -driven β-catenin+ve GFP+ve clusters to publicly available bulk mRNA-seq transcriptomics data sets, including human β-catenin-mutated+ve aCP bulk mRNA-seq data 19 and data from murine models that express a degradation resistant form of β-catenin within Sox2+ve pituitary stem cells ( Sox2 CreERT2/+ ;Ctnnb1 loxP(Exon3)/+ ) 18 ( Fig. 8 b-c ). Gene Ontology (GO) analyses identified common upregulated pathways between our murine Apc -driven model and human β-catenin-mutated+ve aCPs, such as keratinisation, with a high number of keratin and keratin-associated proteins found upregulated ( Fig. 8c & Supplementary Fig. 4 a-b ), inflammatory response, neutrophil chemotaxis, IL2, IL6 and positive regulators of angiogenesis. These pathways are all linked to SASP mechanisms known to occur in β-catenin-mutated+ve aCPs 18-20,49 . One distinction identified in our transcriptomic analyses is that the Apc -driven β-catenin+ve GFP+ve clusters do not upregulate the senescence-associated factor p16 (Cdkn2a), in contrast to the p16 upregulation reported in some human and murine β-catenin-mutated+ve aCP clusters 18 , 19 . Together our data show that Apc -driven β-catenin+ve GFP+ve clusters are transcriptionally analogous, despite some differences, to previously described β-catenin-mutated+ve aCP cell clusters. These clusters exhibit SASP activity and function as secretory hubs within the tumour microenvironment in both murine 7 , 18 and human 19 , 49 aCPs. Download figure Open in new tab Fig 8. Transcriptomic analysis shows that Apc -driven tumours recapitulate human aCPs. a ) Unsupervised Gene Set Enrichment analyses confirm that the β-catenin+ve GFP+ve cell clusters over-activate the Wnt/β-catenin canonical pathway with statistically significant upregulation of IL-2-Stat5, IL6-Stat3 and TNF-α signalling pathways involved in inflammatory response and SASP. The KRAS and p53 pathways were also found upregulated by the β-catenin+ve GFP+ve cell clusters, as were genes involved in epithelial-to-mesenchymal transition. b ) Venn diagram comparing differential gene expression between Cre:Apc LoxP15/15 ; Lef-1:GFP derived clusters and clusters from β-catenin-mutated+ve model ( Sox2 CreERT2/+ ;Ctnnb1 Loxp(Exon3 )/+ ) and from human aCPs reveals higher number of genes in common between human aCP clusters and Apc -driven β-catenin+ve GFP+ve clusters (950 genes in common). c ) Gene Ontology enrichment indicates upregulated pathways in both human aCPs, β-catenin-mutated+ve clusters and our model. These upregulated transcriptional signatures are important in aCP development and include pathways involved with keratinization, inflammasome: neutrophil chemotaxis, interleukin 6 and 2, angiogenesis, the innate immune response, positive regulation of inflammatory response and MAPK pathways. This indicates that the Apc -driven β-catenin+ve GFP+ve clusters are analogous to their β-catenin-mutated+ve aCPs. Abbreviations: GO, Gene Ontology. Discussion Previous genetic studies in mice 6 , 7 and humans 46 , 56 have demonstrated that somatic mutations leading to a non-degradable form of β-catenin are the main driver in a substantial proportion of aCPs. However, the exact prevalence of β-catenin mutations in aCPs varies considerably between published studies 10 - 14 and a significant fraction of aCPs lack a clearly defined driving mutation. Recently, two studies presented an association between mutations in APC leading to FAP in three patients reported to carry germline truncated mutations in APC 31 , 32 . A further possible association between aCPs and either FAP 26 , or APC -pathogenic syndromes such as Gardner 27 and Turcot syndromes 28 has been reported in 8 patients developing aCPs 34 - 43 . However, the lack of full genomic sequencing data for these patients’ tumours has not resolved whether mutations in APC had a direct role in the causation and formation of their aCPs. In this paper, we use three transgenic models to unequivocally show that disruption of Apc in the murine pituitary progenitors/stem cells is a main driver of aCPs independent of mutations in β-catenin. Firstly, we show that biallelic loss of Apc exon15 (Cre:Apc LoxP15/15 ) leads to severe intrauterine aCP tumours, which are not compatible with postnatal life. Importantly, cases of severe intrauterine aCPs have been reported by both our group 8 and others 57 , 58 , indicating that aCPs can present at any stage of life. The severity of the phenotype with lack of all hormone-producing cells, leads to early perinatal lethality in this model. We developed a second model by generating a hypomorphic allele for Apc in which 7.5 % of wild type Apc mRNA is still produced ( Cre:Apc LoxP1-15/15 ) leading to 100% penetrant postnatal aCPs. In this Apc -hypomorphic allele, postnatal tumours develop that have all the histopathological and molecular hallmarks of human aCPs, including large deposits of wet keratin, which were not observed in previously published β-catenin-mutated+ve aCP murine models 6 , 18 . Importantly, this new Apc -driven aCP model, faithfully recapitulates one of the most important aCP-disease comorbidities, tumour-induced morbid obesity, which was not seen in the previous murine model of aCPs driven by oncogenic β-catenin ( Hesx1 Cre/+ ;Ctnnb1 Loxp(Exon3)/+ ) 6 , 18 . Thus, our model represents a robust genetic tool to study aCP tumour-induced hypothalamic obesity, a major complication seen in aCP patients 2,59-62 . It is worth noting that in our Apc -driven aCPs ( Cre:Apc LoxP1- 15/15 ), we observed gender differences in tumour progression with females living longer than males. Whether this reflects a difference in tumour immune microenvironment between genders, as seen in models of Wnt -driven adrenocortical carcinoma (ACCs) 63 d will require further investigation. However, human epidemiological studies from large aCP patient registries do not show gender differences in disease progression for β-catenin-mutated+ve aCPs 2 , 5 , 64 , 65 . Our mouse genetic study provides further information on the role of Apc in tumorigenesis and argues against a dominant negative effect of truncated Apc which has been hypothesised in other cancers 66 , 67 . Our hypomorphic model of aCPs ( Cre:Apc LoxP1-15/15 ) expresses 7.5% of wild type Apc . This is sufficient to rescue the early perinatal lethality observed with bi-allelic loss of Apc exon15 , which is typically associated with more aggressive and larger gestationally derived tumours. Thus, our data show that small amounts of Wt Apc mRNA is sufficient to ameliorate the severe phenotype arguing against a dominant negative role of truncated Apc exon15 . β-catenin-mutated+ve aCPs have been shown to originate from pituitary progenitors/stem cells that express the stem cell marker Sox2 7 . Using a transgenic Sox2 CreERT2/+ to delete Apc exon15 , we show that biallelic loss of Apc exon15 in the Sox2+ve pituitary stem cells is sufficient to produce early aCP lesions indicating that these Apc-tumours arise from progenitor/stem cells of the pituitary gland. Some aCPs are characterised histologically by the presence of nucleo-cytoplasmic β-catenin+ve cell clusters that are a diagnostic hallmark of aCPs 6 , 11 , 17 , 46 . These clusters have been shown to initiate tumour formation and have been studied extensively in β-catenin-mutated+ve aCPs both in mice 6 , 17 , 18 and humans 17 , 19 , 68 . These nucleo-cytoplasmic β-catenin+ve cell clusters exhibit SASP activity, secreting proinflammatory mediators, chemokines, cytokines and growth factors that collectively alter the tumour microenvironment. Our transcriptomic analyses of Apc -driven clusters revealed that, indeed, these tumour-initiating cells undergo SASP and act as secretory hubs to the tumour microenvironment. We identified key secreted SASP factors such as proinflammatory mediators, abundant cytokines and chemokines, interleukins and angiogenesis factors, all of which have been associated with SASP in various tumours 53 - 55 . These clusters secrete numerous downstream targets of the Wnt canonical pathway and developmental growth factors such as Fgfs, Bmps and Shh, which may influence the tumour micro-environment. Indeed, a recent study has shown that in β-catenin-mutated+ve aCPs, clusters can modify tumour-associated macrophages from a M1 (anti-tumorigenic) to M2 (pro-tumorigenic) phenotype in co-culture experiments 68 , an effect that could be similar within Apc- driven tumours. Although, the Apc- driven β-catenin clusters seem largely analogous to their β-catenin-mutated+ve counterparts in humans, some transcriptional differences were noted that could confer specific targeted treatment. Compared to other previously published murine aCP models 6 , 18 , we identify that the Apc -driven clusters express a large number of keratins and keratin-associated proteins which contribute to the large wet-keratin deposits that parallel human aCPs which are absent in other published aCP murine models. Another difference within our Apc -driven tumour model is the mechanism of SASP, which relies on the p21/p53 pathway rather than Cdkn2a (p16), differing from previously published human and murine data on β-catenin-mutated+ve aCPs 17-19,49 . Whether these transcriptional differences are characteristic of all APC-mutated+ve tumours requires additional studies in more aCP samples to enable stratification of transcriptional signatures based on the aCP-mutation specific subtype. Our data call for in-depth genomic studies to identify the overall prevalence of APC -mutations in larger cohorts of patients using high depth of reading sequencing techniques, which will allow for detection of low allelic frequency mutations. These studies will lead to genotype/clinical phenotype correlation in patients without mutations in oncogenic β-catenin and identify whether their clinical trajectories differ i.e. number of recurrences and/or hypothalamic involvement. Importantly, our murine genetic study shows that dysfunction of Apc is a main driver of aCPs independent of mutations in β-catenin and hence APC-mutated+ve tumours represent a novel genetic subtype of aCPs. Since our data demonstrate the causal effect of Apc-dysfunction in the development of aCPs, this highlights the need for genetic testing and increased surveillance of patients with FAP or germline APC -pathogenic syndromes for the development of aCPs. Furthermore, it warrants the inclusion of aCPs as part of the broader spectrum of tumours associated with these syndromic conditions. Equally, patients that develop aCPs with β-catenin-mutated-negative tumours should be tested for underlying germline mutations within APC as a possible cause of FAP or APC-related syndromes such as Gardner and Turcot syndromes. Methods Animal All experiments were conducted under regulations, licenses and local ethical review (QM-AWERB Ethical Committee) following the UK Home Office Animals (Scientific Procedures) Act 1986. The transgenic lines Rosa26 CAGLoxpSTOPLoxpTdTomato (stock #007905) 69 , ApcLoxp(Exon15)/Loxp(Exon15) (stock #029275) 43 , ApcLoxp(Exon1-15)/Loxp(Exon1-15) (stock #009045) 48 , Tcf/Lef-1:H2B-GFP (stock #032838) 51 and the Sox2 CreERT2/+ (stock #017593) 50 were obtained from the JAX-lab and have been previously described. The Prop1:Cre transgenic line 44 , 45 was kindly provided by Shannon Davis and Sally Camper. Animals were kept in 12 h light/12 h dark cycle, with a constant supply of food and water, temperatures of 65–75°F (∼18–23°C) with 40–60% humidity. Kaplan Meier survival curves were plotted using humane end points as determined in line with UK Home Office regulations regarding use of mice in research. Mice were humanly culled when health deterioration was assessed to be irreversible. Tamoxifen induction of the Cre recombinase in embryos ( Sox2 CreERT2/+; Apc LoxP15/15 ) during pregnancy was achieved by 3 consecutive daily injections of 0.1 mg/g of mouse at e12.5, e13.5, e14.5 days of gestation and embryos were dissected at e18.5. Immunohistochemistry, immunofluorescence, and in situ hybridisation Immunostaining was performed as previously described 70 - 72 in short: sections were deparaffinised followed by rehydration through decreasing ethanol dilutions. Heat-induced antigen retrieval was performed with a microwave in 10 mM sodium citrate buffer (pH 6). Samples were left to cool and incubated for 1 h in blocking buffer [1 PBS, 0.1% Triton X-100, 5% Normal Goat Serum (Vector Laboratories)]. Antibodies, their concentration and source are listed in Supplementary Table 1 . Immunohistochemistry (IHC) staining was achieved using DAB Peroxidase Substrate Kit (Vector Laboratories; SK-4100). The colorimetric reaction was stopped with water and sections were counterstained using haematoxylin (Sigma-Aldrich). For immunofluorescence (IF), conjugated secondary antibodies Alexa Fluor 568 or 488 were used, or a biotinylated secondary followed by streptavidin ( Supplementary Table 1 ). Sections were mounted with VECTASHIELD containing DAPI (Vector Laboratories). Images were acquired with a Leica microscope. Figures were generated with Adobe Photoshop CS9. The mitotic index (MI) is the percentage of pHH3-positive cells compared to total number of cells (average counts from three different sections, separated approximately by 100 μm, per embryo/pituitary with a minimum of n = 3–4 per genotype and stage). In situ hybridisation was performed by adapting the protocol from 73 and described before in 72 - 75 . In short, slides were deparaffinised, rehydrated and fixed with 4% PFA. Slides were incubated with proteinase K, followed by a second fixation with 4% PFA and finally incubated with 0.1 M triethanolamine and 0.1% acetic anhydride (Sigma). Hybridisation was achieved by an overnight incubation with 100 ng of the digoxigenin-labelled probe at 65°C. Sections were washed in 0.1 M Tris-HCl Buffer (pH = 7.5) followed by an overnight incubation at 4 °C with anti-Dig antibody (Sigma-Aldrich). Signal detection was achieved by colorimetric reaction using 4-nitro blue tetrazolium chloride solution (NBT; Sigma-Aldrich) and 5-bromo-4-chloro-3-indolyl phosphate disodium salt (BCIP; Sigma-Aldrich). The digoxigenin-labelled antisense probes p21, Axin2, Shh, Fgf8, Fgf3, Wnt6 and Wnt7a were generated from plasmids containing either a portion or full-length cDNA of each respective gene, and plasmids were obtained from Source Bioscience, or gifts from Sally Camper, Andreas Kispert and Peter Gruss and published before 76 - 78 . Pituitary volume quantification was performed using H&E stained pituitaries. Four sections in every 16 were counted for estimation. Microscope images were taken at 5x magnification and analysed using image J software. Estimation of pituitary volume was calculated by multiplying the area of the pituitary by the thickness of each section (7µm) and correcting for the remaining sections in the series [Pituitary volume (µm 3 ) = Measured area(µm 2 ) x 0.7(µm) x 4]. Comparative analyses of pituitary volumes at each embryonic stage from at least 3 different embryos per genotype from different litters were made using Student’s T-Test. Fluorescent activated cell sorting (FACs) To FAC-sort pituitary cell populations, pituitary single cell suspension was achieved by enzymatic treatment using 250 µL enzyme mix [0.05% Collagenase Type II (LorneLaboratories Ltd), 50 µg/ml DNaseI (Worthington), 2.5μg/ml Amphotericin B (Gibco), 0.1% v/v trypsin-EDTA solution 0.05% (Gibco) in Hank’s Balanced Salt Solution (HBSS, Gibco)] in an RNAse and DNAse free 1.5ml microcentrifuge tube (Axygen). Samples were incubated for 1 hour at 37°C under constant agitation at 500 rpm. Pituitaries were mechanically dissociated by repeated pipetting. Dead cells were stained with 500 µL of 1/5000 sterile DAPI solution incubated under gentle agitation for 3 minutes. Samples were centrifuged and cells were resuspended in 500 µL FACS buffer [25 mMHEPES (Gibco), 1% FCS (Sigma-Aldrich) in sterile PBS] by repeated pipetting and transferred to a flow cytometry tube. For mRNA purification, cells were FAC-sorted directly in 750 µL of TRIzolTM-LS (Invitrogen). Cells were sorted using a BD FACs Aria II sorter (BD bioscience) using a A100 µm nozzle. Side scatter (SSCr) area and forward scatter (FSC) area were compared to ensure cellular matter was identified and sorted. SSCr width and FSC area were then compared to ensure only single cells were sorted. A 405 nm laser was used to sort DAPI+ve and DAPI-ve cells. A 561 nm laser was used to sort tdTomato+ve and tdTomato-ve cell populations. A 488 nm laser was used to sort GFP+ve and GFP-ve cells. Sorted cells were immediately snap frozen and stored at -80°C until mRNA purification. RNA purification, RT-qPCR for recombination efficiency and validation of mRNA-seq RNA from FAC-sorted cells from Sox2 CreERT2/+ ;Rosa26 LoxpSTOPLoxPtdTmT , Cre:Apc LoxP15/15 ;Lef-1:GFP or stem cell cultures from Cre:Apc 15/15 or Wt pituitaries was obtained using PureLink™ RNA Micro Kit (Invitrogen) following the manufacture’s protocols. Samples were submitted to quality control using an Agilent 2100 Bioanalyser and only samples with RNA integrity values ≥ 8.1 were kept for mRNA-seq analyses. To generate cDNA for RT-qPCR, 500 ng of RNA was incubated at room temperature for 15 minutes with 1 µL of DNAse I (Invitrogen), 1 µL 10x DNase I Reaction Buffer and UltraPure RNAse free water (Invitrogen) to a total volume of 10 µL. Reverse Transcription was performed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) to produce cDNA according to the manufacturers protocol. Quantity and quality of the cDNA samples were analysed using Nanodrop ND-1000 spectrophotometer (Nanodrop) and quality of cDNA was assessed by determination of the 260/280 and 260/230 ratios. Gene expression levels were analysed by RT-qPCR using QuantiTect SYBR Green PCR Kit (Qiagen) according to the manufacturer’s protocol and analysed with Stratagene (Agilent Technologies). A comparative Ct method (2 −ΔΔCT2 method) was used to compare the mRNA expression levels of genes of interest between cell populations normalised to GAPDH. RT-qPCR primers for GAPDH, Apc exon15, Axin2, Wnt7a, Notum, Fgf3, Ccl2, Ccl3, Gh, Tsh, Pomc, Prl are shown in Supplementary Table 2 . Pituitary stem cells (PSCs) culture and proliferation assays PSCs were cultured from Wt or Cre:Apc loxP15/1-15 mutant pituitaries and incubated for 2 h in enzyme mix [0.5% w/v Collagenase, 50 µg/ml DNase, 2.5 µg/ml Fungizone, trypsin 0.1% in Hank’s Balanced Salt Solution] and mechanically dissociated into single cells. In all, 10,000 cells/well were plated in 12-well plates and cultured in stem cell-promoting media [Ultraculture Medium (Lonza), supplemented with 5% FCS (Sigma), 1% penicillin/streptomycin (P/S: Fisher), 1% glutamax (Fisher), 20 ng/ml basic fibroblast growth factor (R&D) and 50 ng/ml cholera toxin (Sigma)] 72 . Media was changed every 48 h and cultures were maintained for 8 days. Cells were stained with Coomassie blue (Fisher), and either the number of colonies or cells per colony was counted. Statistics and reproducibility Statistical analyses were performed using Prism9 software (GraphPad). The number of independent experiments and replicates ( n ) is indicated in each of the figure legends. Unless stated otherwise, at least three biologically independent replicates were performed for each panel, which came from at least three independent experiments. When appropriate, normalisation of the data was performed within each independent experiment. mRNA-seq and bioinformatic analyses For library preparation mRNA enrichment for cell samples using the SMARTer ultralow input kit (Illumina), and 150bp paired-end sequencing was then performed on a NovaSeq6000 platform. Reads were quality trimmed using Trimgalore v0.6.5 and aligned to reference genome GRCm39 using STAR v2.7.9a 79 . Reads were counted using feature counts, and the ENSEMBL annotation gencode.vM33.annotation.gtf. Downstream analysis was performed in DESeq2 80 , with filtering of genes with less than one read count per million (cpm). Genes with a |logFC|>2 and p.adj 5) and downregulated (logFC< -5) genes separately using the TopGO v2.52.0 package. For unsupervised hierarchical clustering, the top 5000 variable genes (ranked by standard deviation) were used. For comparison to the BcatGOF model 7 and human aCP 19 published data raw fastqs were accessed from the European Nucleotide Archive (PRJEB18056) and analysed as above using GRCh38.release109 and ENSEMBL annotation Homo_sapiens.GRCh38.109.gtf for the human samples. Data availability The authors declare that all the data supporting the findings herein are included in the article (or Supplementary materials) and available from the corresponding author (C.G.M.) upon reasonable request. All sequencing raw data files have been deposited to GEO under reference GSE282683 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE282683 ). The source data underlying graphs and supplementary materials are provided as a Source Data file. A reporting summary for this article is available as a Supplementary Information file. Author Contributions CGM conceived the work, obtained funding, supervised research, performed experiments, analysed data, wrote the manuscript and dealt with all the editorial publishing process. JB, LGC, JN, AR, RT, JX, KCG, AG, PD performed experiments and analysed data. JN, CH, AR bioinformatic analyses. JB, RT & JN contributed to funding acquisition. SWD, provided the Prop1:Cre transgenic line. PD, MK supervised AR JB. FR provided neuropathological expertise. All authors helped with the editing and agreed with its content for publication. Acknowledgements We apologise to the authors whose work we were unable to reference due to space constraints. The research is funded by Action Medical Research (GN 2272), Grants from Barts Charity (GN 417/2238 and MGU0551) and Sparks-GOS (V4323). R.T. and J.B. were funded by Medical Research Council, MRC-CRTF (MR/P018459/1) and (MR/S037896) grants respectively. LGC funded by PhD from Barts Charity (MGU0551). AR was funded by Newton-Bhabha Fund, Academy of Medical Sciences UK (NIFR8\1037). References 1. ↵ Louis , D. N. et al. The 2021 WHO Classification of Tumors of the Central Nervous System: a summary . Neuro Oncol 23 , 1231 – 1251 ( 2021 ). doi: 10.1093/neuonc/noab106 OpenUrl CrossRef PubMed 2. ↵ Muller , H. L. , Merchant , T. E. , Warmuth-Metz , M. , Martinez-Barbera , J. P. & Puget , S. Craniopharyngioma . Nat Rev Dis Primers 5 , 75 ( 2019 ). doi: 10.1038/s41572-019-0125-9 OpenUrl CrossRef PubMed 3. ↵ Brastianos , P. K. et al. Exome sequencing identifies BRAF mutations in papillary craniopharyngiomas . Nat Genet 46 , 161 – 165 ( 2014 ). doi: 10.1038/ng.2868 OpenUrl CrossRef PubMed 4. ↵ Larkin , S. J. , Preda , V. , Karavitaki , N. , Grossman , A. & Ansorge , O. BRAF V600E mutations are characteristic for papillary craniopharyngioma and may coexist with CTNNB1-mutated adamantinomatous craniopharyngioma . Acta Neuropathol 127 , 927 – 929 ( 2014 ). doi: 10.1007/s00401-014-1270-6 OpenUrl CrossRef PubMed 5. ↵ Zacharia , B. E. et al. Incidence, treatment and survival of patients with craniopharyngioma in the surveillance, epidemiology and end results program . Neuro Oncol 14 , 1070 – 1078 ( 2012 ). doi: 10.1093/neuonc/nos142 OpenUrl CrossRef PubMed 6. ↵ Gaston-Massuet , C. et al. Increased Wingless (Wnt) signaling in pituitary progenitor/stem cells gives rise to pituitary tumors in mice and humans . Proc Natl Acad Sci U S A 108 , 1148211487 ( 2011 ). doi: 10.1073/pnas.1101553108 OpenUrl Abstract / FREE Full Text 7. ↵ Andoniadou , C. L. et al. Sox2(+) stem/progenitor cells in the adult mouse pituitary support organ homeostasis and have tumor-inducing potential . Cell Stem Cell 13 , 433 – 445 ( 2013 ). doi: 10.1016/j.stem.2013.07.004 OpenUrl CrossRef PubMed Web of Science 8. ↵ Scagliotti , V. et al. Histopathology and molecular characterisation of intrauterine-diagnosed congenital craniopharyngioma . Pituitary 19 , 50 – 56 ( 2016 ). doi: 10.1007/s11102-015-0682-1 OpenUrl CrossRef PubMed 9. ↵ Karavitaki , N. et al. Craniopharyngiomas in children and adults: systematic analysis of 121 cases with long-term follow-up . Clin Endocrinol (Oxf) 62 , 397 – 409 ( 2005 ). doi: 10.1111/j.1365-2265.2005.02231.x OpenUrl CrossRef PubMed 10. ↵ Hara , T. et al. Clinical and biological significance of adamantinomatous craniopharyngioma with CTNNB1 mutation . J. Neurosurg ., 1 – 10 ( 2018 ). doi: 10.3171/2018.3.JNS172528 OpenUrl CrossRef 11. ↵ Hofmann , B. M. et al. Nuclear beta-catenin accumulation as reliable marker for the differentiation between cystic craniopharyngiomas and rathke cleft cysts: a clinicopathologic approach . Am.J.Surg.Pathol . 30 , 1595 – 1603 ( 2006 ). doi: 10.1097/01.pas.0000213328.64121.12 [doi];00000478-200612000-00014 [pii] OpenUrl CrossRef PubMed Web of Science 12. Marucci , G. et al. Targeted BRAF and CTNNB1 next-generation sequencing allows proper classification of nonadenomatous lesions of the sellar region in samples with limiting amounts of lesional cells . Pituitary 18 , 905 – 911 ( 2015 ). doi: 10.1007/s11102-015-0669-y OpenUrl CrossRef PubMed 13. Apps , J. R. et al. CTNNB1 mutations are clonal in adamantinomatous craniopharyngioma . Neuropathol Appl Neurobiol 46 , 510 – 514 ( 2020 ). doi: 10.1111/nan.12613 OpenUrl CrossRef PubMed 14. ↵ Malgulwar , P. B. et al. Study of beta-catenin and BRAF alterations in adamantinomatous and papillary craniopharyngiomas: mutation analysis with immunohistochemical correlation in 54 cases . J Neurooncol 133 , 487 – 495 ( 2017 ). doi: 10.1007/s11060-017-2465-1 OpenUrl CrossRef PubMed 15. He , J. et al. Characterization of novel CTNNB1 mutation in Craniopharyngioma by whole-genome sequencing . Mol Cancer 20 , 168 ( 2021 ). doi: 10.1186/s12943-021-01468-7 OpenUrl CrossRef PubMed 16. ↵ Wang , X. et al. Multi-omics analysis of adamantinomatous craniopharyngiomas reveals distinct molecular subgroups with prognostic and treatment response significance . Chin Med J (Engl) 137 , 859 – 870 ( 2024 ). doi: 10.1097/CM9.0000000000002774 OpenUrl CrossRef PubMed 17. ↵ Andoniadou , C. L. et al. Identification of novel pathways involved in the pathogenesis of human adamantinomatous craniopharyngioma . Acta Neuropathol 124 , 259 – 271 ( 2012 ). doi: 10.1007/s00401-012-0957-9 OpenUrl CrossRef PubMed Web of Science 18. ↵ Gonzalez-Meljem , J. M. et al. Stem cell senescence drives age-attenuated induction of pituitary tumours in mouse models of paediatric craniopharyngioma . Nat Commun 8 , 1819 ( 2017 ). doi: 10.1038/s41467-017-01992-5 OpenUrl CrossRef PubMed 19. ↵ Apps , J. R. et al. 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 . Acta Neuropathol 135 , 757 – 777 ( 2018 ). doi: 10.1007/s00401-018-1830-2 OpenUrl CrossRef PubMed 20. ↵ Jiang , Y. et al. Single-cell RNA sequencing highlights intratumor heterogeneity and intercellular network featured in adamantinomatous craniopharyngioma . Sci Adv 9 , eadc8933 ( 2023 ). doi: 10.1126/sciadv.adc8933 OpenUrl CrossRef PubMed 21. ↵ Nusse , R. & Clevers , H. Wnt/beta-Catenin Signaling, Disease, and Emerging Therapeutic Modalities . Cell 169 , 985 – 999 ( 2017 ). doi: 10.1016/j.cell.2017.05.016 OpenUrl CrossRef PubMed 22. Clevers , H. , Loh , K. M. & Nusse , R. Stem cell signaling. An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control . Science 346 , 1248012 ( 2014 ). doi: 10.1126/science.1248012 OpenUrl Abstract / FREE Full Text 23. ↵ Iluta , S. , Nistor , M. , Buruiana , S. & Dima , D. Wnt Signaling Pathway in Tumor Biology . Genes (Basel) 15 ( 2024 ). doi: 10.3390/genes15121597 OpenUrl CrossRef 24. ↵ Rubinfeld , B. et al. Binding of GSK3beta to the APC-beta-catenin complex and regulation of complex assembly . Science 272 , 1023 – 1026 ( 1996 ). doi: 10.1126/science.272.5264.1023 OpenUrl Abstract 25. ↵ Klaus , A. & Birchmeier , W. Wnt signalling and its impact on development and cancer . Nat Rev Cancer 8 , 387 – 398 ( 2008 ). doi: 10.1038/nrc2389 OpenUrl CrossRef PubMed Web of Science 26. ↵ Fodde , R. , Smits , R. & Clevers , H. APC, signal transduction and genetic instability in colorectal cancer . Nat Rev Cancer 1 , 55 – 67 ( 2001 ). doi: 10.1038/35094067 OpenUrl CrossRef PubMed Web of Science 27. ↵ Gardner , E. J. & Richards , R. C. Multiple cutaneous and subcutaneous lesions occurring simultaneously with hereditary polyposis and osteomatosis . Am J Hum Genet 5 , 139 – 147 ( 1953 ). OpenUrl PubMed Web of Science 28. ↵ Malbari , F. & Lindsay , H. Genetics of Common Pediatric Brain Tumors . Pediatr Neurol 104 , 3 – 12 ( 2020 ). doi: 10.1016/j.pediatrneurol.2019.08.004 OpenUrl CrossRef PubMed 29. ↵ Foulkes , W. D. A tale of four syndromes: familial adenomatous polyposis, Gardner syndrome, attenuated APC and Turcot syndrome . QJM 88 , 853 – 863 ( 1995 ). OpenUrl PubMed Web of Science 30. ↵ Khattab , A. & Monga , D. K. in StatPearls ( 2025 ). 31. ↵ Gorelyshev , A. et al. Second-hit APC mutation in a familial adamantinomatous craniopharyngioma . Neuro Oncol 22 , 889 – 891 ( 2020 ). doi: 10.1093/neuonc/noaa060 OpenUrl CrossRef PubMed 32. ↵ Passos , J. et al. Familial adenomatous polyposis associated craniopharyngioma secondary to both germline and somatic mutations in the APC gene . Acta Neuropathol 140 , 967 – 969 ( 2020 ). doi: 10.1007/s00401-020-02232-9 OpenUrl CrossRef PubMed 33. ↵ Gabel , B. C. et al. Unusual and Rare Locations for Craniopharyngiomas: Clinical Significance and Review of the Literature . World Neurosurg 98 , 381 – 387 ( 2017 ). doi: 10.1016/j.wneu.2016.10.134 OpenUrl CrossRef PubMed 34. ↵ Aquilina , K. , O’Brien , D. F. , Farrell , M. A. & Bolger , C. Primary cerebellopontine angle craniopharyngioma in a patient with gardner syndrome. Case report and review of the literature . J Neurosurg 105 , 330 – 333 ( 2006 ). doi: 10.3171/jns.2006.105.2.330 OpenUrl CrossRef PubMed 35. Link , M. J. , Driscoll , C. L. & Giannini , C. Isolated, giant cerebellopontine angle craniopharyngioma in a patient with Gardner syndrome: case report . Neurosurgery 51 , 221 – 225 ; discussion 225-226 ( 2002 ). doi: 10.1097/00006123-200207000-00033 OpenUrl CrossRef PubMed 36. Kim , M. S. et al. Primary intracranial ectopic craniopharyngioma in a patient with probable Gardner’s syndrome . J Neurosurg 120 , 337 – 341 ( 2014 ). doi: 10.3171/2013.10.JNS131401 OpenUrl CrossRef PubMed 37. Alvarez Salgado , J. A. et al. [Ectopic craniopharyngioma and Gardner’s syndrome: Case report and literature review] . Neurocirugia (Astur) 28 , 97 – 101 ( 2017 ). doi: 10.1016/j.neucir.2016.09.001 OpenUrl CrossRef PubMed 38. Uemura , H. et al. The association of ectopic craniopharyngioma in the fourth ventricle with familial adenomatous polyposis: illustrative case . J Neurosurg Case Lessons 3 ( 2022 ). doi: 10.3171/CASE21572 OpenUrl CrossRef 39. ↵ Bose , S. et al. Cerebellopontine angle craniopharyngioma in familial adenomatous polyposis . Surg Neurol Int 15 , 340 ( 2024 ). doi: 10.25259/SNI_315_2024 OpenUrl CrossRef PubMed 40. Shah , G. B. , Bhaduri , A. S. & Misra , B. K. Ectopic craniopharyngioma of the fourth ventricle: case report . Surg Neurol 68 , 96 – 98 ( 2007 ). doi: 10.1016/j.surneu.2006.11.073 OpenUrl CrossRef PubMed 41. Powers , C. J. , New , K. C. , McLendon , R. E. , Friedman , A. H. & Fuchs , H. E. Cerebellopontine angle craniopharyngioma: case report and literature review . Pediatr Neurosurg 43 , 158 – 163 ( 2007 ). doi: 10.1159/000098394 OpenUrl CrossRef PubMed 42. ↵ Yan , Y. , Tang , W. Y. , Yang , G. & Zhong , D. Isolated cerebellopontine angle craniopharyngioma . J Clin Neurosci 16 , 1655 – 1657 ( 2009 ). doi: 10.1016/j.jocn.2009.02.029 OpenUrl CrossRef PubMed 43. ↵ Robanus-Maandag , E. C. et al. A new conditional Apc-mutant mouse model for colorectal cancer . Carcinogenesis 31 , 946 – 952 ( 2010 ). doi: 10.1093/carcin/bgq046 OpenUrl CrossRef PubMed 44. ↵ Davis , S. W. , Keisler , J. L. , Perez-Millan , M. I. , Schade , V. & Camper , S. A. All Hormone-Producing Cell Types of the Pituitary Intermediate and Anterior Lobes Derive From Prop1-Expressing Progenitors . Endocrinology 157 , 1385 – 1396 ( 2016 ). doi: 10.1210/en.2015-1862 OpenUrl CrossRef PubMed 45. ↵ Gualtieri , A. et al. Activating mutations in BRAF disrupt the hypothalamo-pituitary axis leading to hypopituitarism in mice and humans . Nat Commun 12 , 2028 ( 2021 ). doi: 10.1038/s41467-021-21712-4 OpenUrl CrossRef PubMed 46. ↵ Buslei , R. et al. Common mutations of beta-catenin in adamantinomatous craniopharyngiomas but not in other tumours originating from the sellar region . Acta Neuropathol 109 , 589 – 597 ( 2005 ). doi: 10.1007/s00401-005-1004-x OpenUrl CrossRef PubMed Web of Science 47. ↵ Garcia-Lavandeira , M. et al. Craniopharyngiomas express embryonic stem cell markers (SOX2, OCT4, KLF4, and SOX9) as pituitary stem cells but do not coexpress RET/GFRA3 receptors . J Clin Endocrinol Metab 97 , E80 – 87 ( 2012 ). doi: 10.1210/jc.2011-2187 OpenUrl CrossRef PubMed Web of Science 48. ↵ Cheung , A. F. et al. Complete deletion of Apc results in severe polyposis in mice . Oncogene 29 , 1857 – 1864 ( 2010 ). doi: 10.1038/onc.2009.457 OpenUrl CrossRef PubMed Web of Science 49. ↵ Prince , E. W. et al. Unraveling the complexity of the senescence-associated secretory phenotype in adamantinomatous craniopharyngioma using multimodal machine learning analysis . Neuro Oncol 26 , 1109 – 1123 ( 2024 ). doi: 10.1093/neuonc/noae015 OpenUrl CrossRef 50. ↵ Arnold , K. et al. Sox2(+) adult stem and progenitor cells are important for tissue regeneration and survival of mice . Cell Stem Cell 9 , 317 – 329 ( 2011 ). doi: 10.1016/j.stem.2011.09.001 OpenUrl CrossRef PubMed Web of Science 51. ↵ Ferrer-Vaquer , A. et al. A sensitive and bright single-cell resolution live imaging reporter of Wnt/ss-catenin signaling in the mouse . BMC Dev Biol 10 , 121 ( 2010 ). doi: 10.1186/1471-213X-10-121 OpenUrl CrossRef PubMed 52. ↵ Liu , B. et al. Regulation of cellular senescence in tumor progression and therapeutic targeting: mechanisms and pathways . Mol Cancer 24 , 106 ( 2025 ). doi: 10.1186/s12943-025-02284-z OpenUrl CrossRef PubMed 53. ↵ Ohtani , N. The roles and mechanisms of senescence-associated secretory phenotype (SASP): can it be controlled by senolysis? Inflamm Regen 42 , 11 ( 2022 ). doi: 10.1186/s41232-022-00197-8 OpenUrl CrossRef 54. Meguro , S. & Nakanishi , M. Cellular senescence in the cancer microenvironment . J Biochem 177 , 171 – 176 ( 2025 ). doi: 10.1093/jb/mvaf001 OpenUrl CrossRef PubMed 55. ↵ Schmitt , C. A. , Wang , B. & Demaria , M. Senescence and cancer - role and therapeutic opportunities . Nat Rev Clin Oncol 19 , 619 – 636 ( 2022 ). doi: 10.1038/s41571-022-00668-4 OpenUrl CrossRef PubMed 56. ↵ Sekine , S. et al. Craniopharyngiomas of adamantinomatous type harbor beta-catenin gene mutations . Am J Pathol 161 , 1997 – 2001 ( 2002 ). doi: 10.1016/s0002-9440(10)64477-x OpenUrl CrossRef PubMed Web of Science 57. ↵ Chentli , F. , Belhimer , F. , Kessaci , F. & Mansouri , B. Congenital craniopharyngioma: a case report and literature review . J Pediatr Endocrinol Metab 25 , 1181 – 1183 ( 2012 ). doi: 10.1515/jpem-2012-0251 OpenUrl CrossRef 58. ↵ do Prado Aguiar , U., Araujo , J. L. , Veiga , J. C. , Toita , M. H. & de Aguiar , G. B. Congenital giant craniopharyngioma . Childs Nerv Syst 29 , 153 – 157 ( 2013 ). doi: 10.1007/s00381-012-1919-1 OpenUrl CrossRef PubMed 59. Erfurth , E. M. & Muller , H. L. Metabolic complications and their mechanisms in patients with craniopharyngioma . Best Pract Res Clin Endocrinol Metab, 101999 ( 2025 ). doi: 10.1016/j.beem.2025.101999 OpenUrl CrossRef 60. Muller , H. L. Long-term quality of life and hypothalamic dysfunction after craniopharyngioma . J Neurooncol ( 2025 ). doi: 10.1007/s11060-025-04987-1 OpenUrl CrossRef 61. Apps , J. R. , Muller , H. L. , Hankinson , T. C. , Yock , T. I. & Martinez-Barbera , J. P. Contemporary Biological Insights and Clinical Management of Craniopharyngioma . Endocr Rev 44 , 518 – 538 ( 2023 ). doi: 10.1210/endrev/bnac035 OpenUrl CrossRef PubMed 62. Muller , H. L. et al. Hypothalamic syndrome . Nat Rev Dis Primers 8 , 24 ( 2022 ). doi: 10.1038/s41572-022-00351-z OpenUrl CrossRef PubMed 63. ↵ Wilmouth , J. J. , Jr. et al. Sexually dimorphic activation of innate antitumor immunity prevents adrenocortical carcinoma development . Sci Adv 8 , eadd0422 ( 2022 ). doi: 10.1126/sciadv.add0422 OpenUrl CrossRef PubMed 64. ↵ Muller , H. L. Craniopharyngioma . Endocr Rev 35 , 513 – 543 ( 2014 ). doi: 10.1210/er.2013-1115 OpenUrl CrossRef PubMed Web of Science 65. ↵ Momin , A. A. et al. Descriptive epidemiology of craniopharyngiomas in the United States . Pituitary 24 , 517 – 522 ( 2021 ). doi: 10.1007/s11102-021-01127-6 OpenUrl CrossRef PubMed 66. ↵ Zhang , L. et al. Selective targeting of mutant adenomatous polyposis coli (APC) in colorectal cancer . Sci Transl Med 8 , 361ra140 ( 2016 ). doi: 10.1126/scitranslmed.aaf8127 OpenUrl Abstract / FREE Full Text 67. ↵ Su , L. K. et al. Association between wild type and mutant APC gene products . Cancer Res 53 , 2728 – 2731 ( 1993 ). OpenUrl Abstract / FREE Full Text 68. ↵ Wang , X. et al. Single-cell and spatial sequencing identifies senescent and germinal tumor cells in adamantinomatous craniopharyngiomas . Cell Biosci 14 , 112 ( 2024 ). doi: 10.1186/s13578-024-01299-1 OpenUrl CrossRef PubMed 69. ↵ Madisen , L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain . Nat Neurosci 13 , 133 – 140 ( 2010 ). doi: 10.1038/nn.2467 OpenUrl CrossRef PubMed Web of Science 70. ↵ Gaston-Massuet , C. et al. Genetic interaction between the homeobox transcription factors HESX1 and SIX3 is required for normal pituitary development . Dev Biol 324 , 322 – 333 ( 2008 ). doi: 10.1016/j.ydbio.2008.08.008 OpenUrl CrossRef PubMed Web of Science 71. Rai , A. et al. Phosphorylation of beta-catenin at Serine552 correlates with invasion and recurrence of non-functioning pituitary neuroendocrine tumours . Acta Neuropathol Commun 10 , 138 ( 2022 ). doi: 10.1186/s40478-022-01441-5 OpenUrl CrossRef PubMed 72. ↵ Gaston-Massuet , C. et al. Transcription factor 7-like 1 is involved in hypothalamo-pituitary axis development in mice and humans . Proc Natl Acad Sci U S A 113 , E548 – 557 ( 2016 ). doi: 10.1073/pnas.1503346113 OpenUrl Abstract / FREE Full Text 73. ↵ Moorman , A. F. , Houweling , A. C. , de Boer , P. A. & Christoffels , V. M. Sensitive nonradioactive detection of mRNA in tissue sections: novel application of the whole-mount in situ hybridization protocol . J Histochem Cytochem 49 , 1 – 8 ( 2001 ). doi: 10.1177/002215540104900101 OpenUrl CrossRef PubMed Web of Science 74. Giri , D. et al. Novel FOXA2 mutation causes Hyperinsulinism, Hypopituitarism with Craniofacial and Endoderm-derived organ abnormalities . Hum Mol Genet 26 , 4315 – 4326 ( 2017 ). doi: 10.1093/hmg/ddx318 OpenUrl CrossRef PubMed 75. ↵ Gaston-Massuet , C. , Henderson , D. J. , Greene , N. D. & Copp , A. J. Zic4, a zinc-finger transcription factor, is expressed in the developing mouse nervous system . Dev Dyn 233 , 1110 – 1115 ( 2005 ). doi: 10.1002/dvdy.20417 OpenUrl CrossRef PubMed Web of Science 76. ↵ Potok , M. A. et al. WNT signaling affects gene expression in the ventral diencephalon and pituitary gland growth . Dev Dyn 237 , 1006 – 1020 ( 2008 ). doi: 10.1002/dvdy.21511 OpenUrl CrossRef PubMed Web of Science 77. Trowe , M. O. et al. Inhibition of Sox2-dependent activation of Shh in the ventral diencephalon by Tbx3 is required for formation of the neurohypophysis . Development 140 , 2299 – 2309 ( 2013 ). doi: 10.1242/dev.094524 OpenUrl Abstract / FREE Full Text 78. ↵ Walther , C. et al. Pax: a murine multigene family of paired box-containing genes . Genomics 11 , 424 – 434 ( 1991 ). doi: 10.1016/0888-7543(91)90151-4 OpenUrl CrossRef PubMed Web of Science 79. ↵ Dobin , A. et al. STAR: ultrafast universal RNA-seq aligner . Bioinformatics 29 , 15 – 21 ( 2013 ). doi: 10.1093/bioinformatics/bts635 OpenUrl CrossRef PubMed Web of Science 80. ↵ Love , M. I. , Huber , W. & Anders , S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 . Genome Biol 15 , 550 ( 2014 ). doi: 10.1186/s13059-014-0550-8 OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted September 29, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Disruption of the Wnt-antagonist Apc in the pituitary stem cells drives the development of adamantinomatous craniopharyngioma Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Disruption of the Wnt-antagonist Apc in the pituitary stem cells drives the development of adamantinomatous craniopharyngioma James Blackburn , Laura Gomez-Corral , James Nicholson , Ashutosh Rai , Jingyi Xue , Rachael Tan , Kalyana C. Gangaraju , Angelica Gualtieri , Charlotte Hall , Pinaki Dutta , Márta Korbonits , Federico Roncaroli , Shannon W Davis , Carles Gaston-Massuet bioRxiv 2025.09.28.679021; doi: https://doi.org/10.1101/2025.09.28.679021 Share This Article: Copy Citation Tools Disruption of the Wnt-antagonist Apc in the pituitary stem cells drives the development of adamantinomatous craniopharyngioma James Blackburn , Laura Gomez-Corral , James Nicholson , Ashutosh Rai , Jingyi Xue , Rachael Tan , Kalyana C. Gangaraju , Angelica Gualtieri , Charlotte Hall , Pinaki Dutta , Márta Korbonits , Federico Roncaroli , Shannon W Davis , Carles Gaston-Massuet bioRxiv 2025.09.28.679021; doi: https://doi.org/10.1101/2025.09.28.679021 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Cancer Biology Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17691) Bioengineering (13892) Bioinformatics (41936) Biophysics (21452) Cancer Biology (18588) Cell Biology (25504) Clinical Trials (138) Developmental Biology (13378) Ecology (19899) Epidemiology (2067) Evolutionary Biology (24320) Genetics (15609) Genomics (22506) Immunology (17736) Microbiology (40394) Molecular Biology (17181) Neuroscience (88605) Paleontology (666) Pathology (2832) Pharmacology and Toxicology (4824) Physiology (7641) Plant Biology (15153) Scientific Communication and Education (2045) Synthetic Biology (4294) Systems Biology (9825) Zoology (2271)