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M Bujko, P Kober and Transcriptomic profiling in 181:6 615–627 Clinical Study others corticotrophinomas

USP8 mutations in corticotroph adenomas determine a distinct gene expression profile irrespective of functional tumour status

Mateusz Bujko1,*, Paulina Kober1,*, Joanna Boresowicz1, Natalia Rusetska1, Agnieszka Paziewska2,6, Michalina Dąbrowska2, Agata Piaścik3, Monika Pękul3, Grzegorz Zieliński4, Jacek Kunicki5, Wiesław Bonicki5, Jerzy Ostrowski2,6, Janusz A Siedlecki1 and Maria Maksymowicz3

1Department of Molecular and Translational Oncology, 2Department of Genetics, 3Department of Pathology and Laboratory Diagnostics, Maria Sklodowska-Curie Institute – Oncology Center, Warsaw, Poland, 4Department of Neurosurgery, Military Institute of Medicine, Warsaw, Poland, 5Department of Neurosurgery, Maria Sklodowska-Curie Institute – Correspondence Oncology Center, Warsaw, Poland, and 6Department of Gastroenterology, Hepatology and Clinical Oncology, Medical should be addressed Center for Postgraduate Education, Warsaw, Poland to M Maksymowicz *(M Bujko and P Kober contributed equally to this work) Email [email protected]

Abstract

Objective: Pituitary corticotroph adenomas commonly cause Cushing’s disease (CD) but part of these tumours are hormonally inactive (silent corticotroph adenomas, SCA). USP8 mutations are well-known driver mutations in corticotrophinomas. Differences in transcriptomic profiles between functioning and silent tumours or tumours with differentUSP8 status have not been investigated. Design and methods: Forty-eight patients (28 CD, 20 SCA) were screened for USP8 mutations with Sanger sequencing. Twenty-four patients were included in transcriptomic profiling with Ampliseq Transcriptome Human Gene Expression Core Panel. The entire patients group was included in qRT-PCR analysis of selected genes expression. Immunohistochemistry was used for visualization of selected protein.

European Journal of Endocrinology Results: We found USP8 mutation in 15 patients with CD and 4 SCAs. USP8 mutations determine molecular profile of the tumours as showed by hierarchical clustering and identification of 1648 genes differentially expressed inUSP8 - mutated and USP8-wild-type tumours. Mutations affect many molecular pathways as observed in Gene Set Enrichment analysis. USP8-mutated adenomas showed higher level of POMC, CDC25A, MAPK4 but lower level of CCND2, CDK6, CDKN1B than USP8-wt tumours. Eighty-seven genes differentially expressed between CD-related adenomas and SCAs were found, including those involved in cell signalling (GLI2, DLC1, TBX2, RASSF6), cell adhesion (GJA1, CDH6), ion transport (KCNN4, KCNJ5) and GABA signalling (GABBR2, GABRD). Conclusion: USP8 mutations occur in functioning and silent corticotrophinomas. They have pleiotropic effect, not limited to EGFR signalling, and affect expression levels of many genes involved in different pathways. Expression of GABA-related genes GABBR2, GNAL, GABARD and KCNJ5 correspond to functional status of the tumours.

European Journal of Endocrinology (2019) 181, 615–627

Introduction

Pituitary tumours represent about 10–20% of all tumours may arise from different kinds of secretory cells intracranial neoplasms and over 90% of tumours are of pituitary gland, and therefore, they are characterized located in the sella turcica; their prevalence in the by overproduction of certain hormones which determine population is estimated to be approximately 1/1000. These the clinical manifestation of the disease.

https://eje.bioscientifica.com © 2019 European Society of Endocrinology Published by Bioscientifica Ltd. https://doi.org/10.1530/EJE-19-0194 Printed in Great Britain Downloaded from Bioscientifica.com at 09/25/2021 12:53:01AM via free access

-19-0194 European Journal of Endocrinology https://eje.bioscientifica.com reevaluated histopathologically. samples ofSCAfromyears2013 to2017werechosenand including 28samplesfrom patientswithCDand20 on WHO2004criteria.Archival FFPEtissuesamples, for routinediagnosticprocedures. Diagnoseswerebased andfixed informalin during transsphenoidalsurgery tumoursamplesfrom48patients werecollected Pituitary Patients andsamples Subjects andmethods mutations affecttranscriptomicprofilesofCDandSCA. in SCAs.Moreover, weintendedtoverifyhow we intended to verify whether its mutations occur also gene mutatedinlargeproportionofcorticotrophinomas, activities.Since secretory biological mechanism contributingto distinct hormonal profiles offunctioningandSCAsinordertoidentify a molecular techniquesislacking. on SCAsandCD-causingtumoursusinggenome-wide poorlyunderstood( is very the molecularbasisofaggressivegrowththesetumours field werepublished( fewstudiesinthis is stillunclearandtheresultsofvery electron microscopy)remainendocrinologicallyinactive and adenomas (asconfirmedbyimmunohistochemistry unexplored. ThereasonwhysomeACTH-producing ( ofrecurrent the discovery molecular backgroundofCDhasbeenmade( characterized byinvasivegrowthandhighrelapserate( tumours of themostaggressivesubtypespituitary diagnosed atmacroadenomastageandrepresentone immunohistochemical staining.SCAsarecommonly confirmed inhistopathologicalexaminationbasedon size ( visual impairment)whentumoursreachmacroadenoma are diagnosedduetoneurologicalsymptoms(forexample adenomas(NFPAs),non-functioning pituitary SCApatients corticotroph adenomas(SCAs).Similartomostpatientswith do notcauseCDsymptomsandareclassifiedassilent functioning ( cellsarehormonallynon- from corticotrophicpituitary disorder. However, aproportionoftumoursoriginating dependent Cushing’s disease(CD)asevere,life-threatening 3 , Clinical Study 4 The aimofthestudywastocomparegeneexpression Recently, notableprogressintheunderstandingof Corticotroph adenomascommonlycauseACTH- , > 5 10 ). Incontrary, thebiologyofSCAstillremains mm). Corticotrophic nature of the tumours is mm). Corticotrophicnatureofthetumoursis 1 ). They do not affect hormonal balance, ). Theydonotaffecthormonalbalance, 6 , 7 , USP8 8 , 14 9 USP8 , , isawell-recognizeddriver 10 15 others M Bujko,PKoberand , , hotspotmutation 11 16 , , 12 17 , ) andresearch 13 2 ). Similarly, ) including USP8 1 ).

International, Gif-sur-Yvette Cedex,France).Theanalytical levels wereassessedusingIRMA(ELSA-ACTH,CISBio tests.ACTH and symptomsaswellbasiclaboratory any evidenceofhypercortisolemia basedonclinicalsigns imaging. InthegroupofSCAs,nonepatientshad (100 of acorticotrophin-releasinghormonestimulationtest suppression test(2 UFC suppression of CDwasconfirmedbasedontheserumcortisollevelsor suppression test(1 < and alackofsuppressionserumcortisollevelsto increased ornotsuppressedplasmaACTHlevelsat8:00 rhythm, increasedserumcortisollevelsaccompaniedby 24-h urinecollections,disturbancesofcortisolcircadian freecortisol(UFC)inthree criteria: increasedurinary hypercortisolemia wasbasedonstandardhormonal Biosystems). ThePCRproduct waspurifiedusingExoStar Diagnostics) using GeneAmp 9700 PCRsystem(Applied PCR amplifiedwithFastStart Taq DNAPolymerase(Roche 14) was determined using Sanger sequencing. DNA was The presenceofpointmutation at Assessment of were storedat NanoDrop 2000(ThermoFisherScientific).Thesamples FFPE (ThermoFisherScientific)andmeasuredusing using RecoverAll™Total NucleicAcid Isolation Kitfor data supplementary Tablein detailSupplementary 1(seesectionon ranged between80 and 100%(median99%),asdescribed The content of tumour tissue in each FFPE sample purposes. consent fortheuseoftissuesamplesscientific Center inWarsaw, Poland.Eachpatientprovidedinformed Committee ofMariaSklodowska-CurieInstitute–Oncology Table 1 reference range:4.3–176 immunoassay (Elecsys 2010, Roche Diagnostics) – with dichloromethane) by electrochemiluminescence UFC wasdeterminedafterextraction(liquid/liquid assay was0.02 (Roche Diagnostics).Analyticalsensitivityofthe Elecsys 2010electrochemiluminescenceimmunoassay Serum cortisolconcentrationsweredeterminedbythe sensitivity was2 corticotrophinomas Transcriptomic profilingin 1.8 The diagnosisofCushing’s syndrome/ DNA and total RNA from FFPE samples was isolated Characteristics ofthepatientsarepresentedin mg i.v.) magneticresonance andpositivepituitary µg/dL duringanovernightdexamethasone . The study was approved by the Local Ethics . ThestudywasapprovedbytheLocalEthics − 70°C. μ USP8 g/dL (referencerange:6.2–19.4 pg/mL (referencerange:10–60 < mg at midnight). The pituitary aetiology mg atmidnight).Thepituitary 50% withahigh-dosedexamethasone mg q.i.d.for48 givenattheendofthisarticle). mutationstatus Downloaded fromBioscientifica.com at09/25/202112:53:01AM μ g/24 h. h) orapositiveresult USP8 181 :6 hotspot(exon pg/mL). μ g/dL). 616 via freeaccess h,

European Journal of Endocrinology Particles, followed by particle recovery andtemplate Particles, followedbyparticle recovery Instrument, whichperforms emulsionPCRonIonSphere preparation withtheIonPIHi-Q ChefKitontheIon barcoded librariesweresubjectedtoautomatedtemplate to ~55 was diluted a Bioanalyzer2100(Agilent).Eachlibrary using AgilentHighSensitivityDNAAnalysisKitson was determined and DNAfragment length ofeach library stored at Agencourt AMPure XPbeads(Beckman Coulter) and the amplicons. The samples were then purified using amplicons weredigestedandadaptersligated to Panel (ThermoFisherScientific).Primersequences of AmpliSeq Transcriptome Human Gene Expression Core RNA wassubjectedtomultiplexamplificationwithIon to themanufacturer’s protocol.Briefly, 100 Gene ExpressionKit(ThermoFisherScientific)according prepared usingtheIonAmpliSeq™Transcriptome Human was sequencing. Human transcriptome sequencing library preparationandionsemiconductor based library RNA isolatedfromFFPEsampleswasusedforamplicon- next-generation sequencing High-throughput transcriptomeprofilingwith (NP_005145.3) sequencewasusedasreference. Tableare presented in Supplementary 2. NM_005154.5 Analyzer (Applied Biosystems). PCR primers sequences electrophoresis withtheABIPRISM3300Genetic manufacturer’s instructionsandanalysedbycapillary Terminator v.3.1 (AppliedBiosytems)accordingtothe (GE HealthcareLifeSciences),labelledwithBigDye USP8 Microadenoma Macroadenoma Tumour size 4 3 2 1 0 KNOSP grade Densely granulated Sparsely granulated Ultrastructural characteristics Female Male Gender Age (years;median(range)) Number ofpatients Table 1 Clinical Study mutation pM priortotemplatepreparation. Uptoseven Patients’ characteristics. − 20°C forfurtherprocessing.Theconcentration 43 (23–76) others M Bujko,PKoberand 11 11 17 12 19 25 28 CD 4 3 4 5 9 3 ng oftotal 53 (23–77) 20 12 11 20 SCA 4 0 2 2 5 9 2 8 9 (FC), calculatedasreadcountsratio models wasperformed.Foldchangeofexpression distributions and negative binomial generalized linear expressed genesbetweengroupsbyuseofdiscrete included) and subsequent analysis of differentially sequencing readsinatleasthalfofthesampleswere gene filtrationwasapplied(thegeneswithatleastfive estimates of dispersion ( normalizationandcalculated performed thenecessary package ( generated from BAM files using Genomic Alignments environment. Therawun-normalizedcountmatrixwas Bioconductor packagesinRprogramminglanguage manufacturer’s instructions(ThermoFisherScientific). as partoftheIonPI™Hi-Q™ChefKitaccordingto reagents (IonPI™Hi-Q™Sequencing200Kit)provided Proton instrumentonaPIchipusingthesequencing loading on a PI chip. Samples were sequenced in the Ion 4- Immunohistochemical staining (IHC)wasperformedon Immunohistochemical staining of calculation ofrelativegeneexpressionlevelwiththeuse Tablein Supplementary 2. PCRnondetectswereusedin expression level.PCRprimers’sequencesarepresented efficiency. DeltaCtmethodwasusedtocalculate relative template concentrations,wereusedfordeterminingPCR basedonamplificationofknown cDNA Standard curves, amplified intriplicates. containing 2.25 Mix (Thermo Fisher Scientific) inavolume of5 (Applied Biosystems)withPowerSYBRGreenPCRMaster format andthe7900HTFastReal-Time PCRSystem Diagnostics). qRT-PCR wascarriedoutusinga384-well the Transcriptor FirstStrandcDNASynthesisKit(Roche RNA samplewassubjectedtoreversetranscriptionusing qRT-PCR. OnemicrogramofeachFFPEtissue-derived Expression levelsoftheselectedgeneswereassessedusing Gene expressionassessmentusingqRT-PCR analysis (GSEa). FC defined asthosewithadjusted of samples.Differentiallyexpressedgenes(DEGs)were as ameasureofexpressiondifferencebetweenthegroups ( corticotrophinomas Transcriptomic profilingin USP8 μ nondetects m FFPEtissuesectionsfrom 24samples(12SCAand > All thesteps of dataanalysis were performed using 2.5 orFC mut)/ 18 USP8 ). ThenitwasimportedtoDESeq2which Rpackage( < pmol ofeachprimer. Thesampleswere -wild type( 0.4 wereusedforGeneSetEnrichment GAPDH 20 Downloaded fromBioscientifica.com at09/25/202112:53:01AM 19 ). USP8 ), and then low-expression wasusedasreferencegene. P https://eje.bioscientifica.com value wt) orCD/SCAwasused 181 < :6 0.05. DEGswith USP8 -mutated 617 µL, via freeaccess European Journal of Endocrinology https://eje.bioscientifica.com (one patient),p.Ser718Pro (two patients)andin-frame variants: p.Pro720Arg(five patients),p.Pro720Gln and 4/20(20%)SCAs.The mutationsweremissense 15 patientsincluding11/28 (39.2%)ofCDadenomas Hot spotmutationsinexon 14of Prevalence of Results used forheatmappreparation. University SchoolofMedicine,Stanford,CA,USA)were enriched. Cluster 3.0 and TreeView 1.6 software (Stanford of adjusted (GSE) analysis ( was used.EnrichRtoolusedforGeneSetEnrichment clustering with Euclidean distance as similarity metrics hclust function,(statsRpackage).Complete-linkage all thegenesthatpassedfiltrationwasdoneusing Software). Hierarchical clustering using the values for analysed andvisualizedusingGraphPadPrism(GraphPad significance thresholdlevel exact testwasusedfortheanalysisofproportions.A the two-sided Mann–Whitney Quantitative continuousvariableswereanalysedusing Statistical analysis results wereanalysedascontinuousvariables. reported formulawasusedforquantification( and numberofthecellswithagivenintensity. Previously information onbothreactionintensity(scoredfrom0to3) performed by calculating H-score, which combines Analysis ofimmunohistochemicalreactivitywas contrast wasachievedwithhematoxylincounterstaining. tetrahydrochloride (Dako) asasubstrate, and nuclear product wasdevelopedwith3,3 the EnvisionDetectionSystem(Dako).Thecolourreaction Scientific) (dilution 1:50) and subsequently labelled with antibody anti-KCNJ5(PA5-53594, ThermoFisher Slides wereincubatedovernightat4°Cwithpolyclonal 5 for 25 water bath,for20 out in a Target Retrieval Solution pH 9 (Dako) in a 96°C concentration. Heat-inducedepitoperetrievalwascarried rehydrated inaseriesofethanolsolutionsdecreasing (Dako). Sectionsweredeparaffinizedwithxyleneand 12 CDtumours)usingtheEnvisionDetectionSystem min withaBlockerofEndogenousPeroxidase(Dako). Clinical Study min atroomtemperature,theslidesweretreatedfor P

< 22 USP8

0.05 were considered assignificantly min. Aftercoolingtheretrievalsolutions ). The GSE terms meeting the criterion mutationinpatients α

= 0.05 wasused.Datawere U others M Bujko,PKoberand test.Two-sided Fisher’s USP8 ′ -diaminobenzidine werefoundin 21 ). Scoring USP8 Twenty-four patientsincluding12CD(6 Effect of (mean age43.8vs50.8,respectively; in ageofthe in 1/12manand14/36women; P granulated andin5/21ofsparselyadenomas, tumours ( according to resultsof ultrastructural examination of the of threshold level( patients, butthedifferencedidnotreachsignificance prevalence ofmutationswashigherinCDthanSCA Fig.1.Theobserved are presentedinSupplementary were heterozygous.TheresultsofSangersequencing deletion p.Ser718del(sevenpatients).Allthemutations organization, cell-celladhesion andregulationofcell system developmentand functioning, nucleosome with DEGsrelatedtofew generalprocesses:nervous by GSEanalysis.We identified11GOtermsenriched the molecular pathways represented by these 595 DEGs Pathways databaseswithEnrichRtool( Ontology (GO)analysisaswellPantherandReactome 1B 595 geneswithFC tumours (adjusted of 1648DEGsdifferentiating corticotroph adenomaswasconfirmedbyidentification role of their endocrinological status ( represent aseparategeneexpressioncluster, irrespectiveof transcriptional datarevealedthat GSE132982 accessionnumber. Gene ExpressionOmnibusandareaccessiblethrough avoid technicalbias.ThedataweredepositedinNCBI's 2). Thesesampleswereexcludedfromtheanalysisto Fig. size showed four outlier samples (Supplementary analysis performedonreadcountsnormalizedforlibrary well as between between functioningandsilentcorticotrophinomasas and identification of genes differentially expressed subjected toanalysisthatinvolvedhierarchical clustering filtration andnormalization,17550humangeneswere Human GeneExpressionCorePanel.Followingdata profiling withtheuseofIonAmpliSeq Transcriptome USP8 corticotrophinomas Transcriptomic profilingin

= and listed in Supplementary Table andlistedinSupplementary 3.UsingGene 0.366) andsexofthepatients(mutationwasdetected USP8 Hierarchical clustering of samplesbasedonwhole mut and6 mut and8 mutations was observed inpatientsstratified mutationswasobserved USP8 USP8 USP8 mutationsindefiningthephenotypeof USP8 mutation was detected in 10/27 of densely mutationongeneexpressionprofile USP8 P

USP8 = USP8 0.2122). Nodifferenceinthefrequency P > mut and value wt) weresubjectedtogeneexpression 2.5 orFC wt) aswell12SCApatients(4 mut and Downloaded fromBioscientifica.com at09/25/202112:53:01AM < 0.05). TheseDEGs,including USP8 Fig. 1A < P 0.4, arepresentedin .79. o difference No =0.0729). USP8 USP8 wt patientswasfound 181 P ). The predominant USP8 =0.1220). mut and wt tumours. PCA :6 22 mut tumours ), wesought USP8 618 Fig. wt via freeaccess European Journal of Endocrinology genes were found (Supplementary Tablegenes werefound(Supplementary 4)including66 corticotrophinomas, eighty-seven differentially expressed When comparingsilent andfunctional silent corticotrophinomas Gene expressioninfunctioningand Tabledetails areshowninSupplementary 3. tumours. Top GSEresultsarepresentedin that areexpressedatsignificantlylowerlevelin of genes encoding replication-dependent histone variants pathways wereidentifiedduetothehighrepresentation were identifiedassignificantlyenriched.Mostof the signalling pathways.Seventy-threereactomepathways as significantlyenriched:cadherinsignallingandWnt proliferation. Two Panther pathways were identified of genesdifferentiallyexpressedinSCAsandadenomascausingCushing’sdiseasewithFC DEGs identifiedincomparisonsof clustering oftumoursamplesbasedonthetranscriptionalprofile(17550genes).(B)Volcanoplotspresentingdistribution of Results oftranscriptomicprofilingcorticotrophtumourswithregardsto Figure 1 Clinical Study AB USP8 others M Bujko,PKoberand

−Log10 P −Log10 P 10 mut vs 10 15 20 0 5 0 5 USP Differentially expressedgene Differentially expressedgene Silent −5.0 −5.0 8 USP8 Table 2 mut vs. vs.f wt tumoursaswellSCAsvsfunctioningcorticotrophinomas.(C)Expression FC<0.4 FC<0.4 USP8 −2.5 −2.5 unctioning , while USP mut Log 8 wt corticotrophinomas 2 fo 0.02 0.02 ld chang corticotrophinomas GABBR2 Table 4).Three DEGswereannotatedtothesepathways: for others. Thehighestfold change ofexpressionwas observed channels functioning( PDGFRB signalling ( play a role in different cellular processes including cell 2B genes withFC differentially expressedgenes ( 977444) astheonlypathway significantlyenrichedforthe activation (ReactomepathwaysR-HSA-991365andR-HSA- analysis withReactomedatabaseshowedGABABreceptor GABAB receptorpathwayaccordingtoReactomedb.GSE downregulated in SCAs) genes that both belong to a corticotrophinomas Transcriptomic profilingin e . These genes encode functionally diverse proteins that KCNJ5 FC>2.5 FC>2.5 USP8 .5 .5 , , Adjusted p=0.05 KCNJ5 Adjusted p=0.05 (mostupregulatedinSCAs)and GABBR2 GLI2 mutationandfunctionalstatus.(A)Hierarchical > and 5.0 , 5.0 2.5 orFC DLC1 ), celladhesion( GNAL > USP8 status C 2.5 orFC Diagnosis , Downloaded fromBioscientifica.com at09/25/202112:53:01AM TBX2 KCNN4 . Nosignificantenrichment was < 0.4 thatarepresentedin , Table 2 < RASSF6 https://eje.bioscientifica.com , 0.4. Diagnosis KCNJ5 CD 181 and Supplementary andSupplementary GJA1 , SCA :6 GNAL , GABBR2 , USP8 status GABRD wt CDH6 , RASGRP2 mut (most ) and ), ion 619 Fig. via freeaccess , European Journal of Endocrinology https://eje.bioscientifica.com CDC25A USP8 selected genesidentified as differentiallyexpressedin Human Gene Expression Core Panel, POMC adenoma tumoursampleswithqRT-PCR. This setincluded The expressionlevelsof12geneswereassessedin48 genes withqRT-PCR Evaluation oftheexpressionlevelselected and Pantherpathways. found inGSEanalysisbasedonGObiologicalprocesses Up totoptenpathwaysarepresented. 2 1 Reactome pathways GSE analysisforgenesdifferentiallyexpressedinSCAsandfunctioningcorticotrophinomas 10 9 8 7 6 5 4 3 2 1 Reactome pathways 2 1 Panther pathways 10 9 8 7 6 5 4 3 2 1 GO molecularprocesses GSE analysisforgenesdifferentiallyexpressedinUSP8-mutatedandUSP8wtpatients tumours. Table 2 Clinical Study mut and genenotincludedinIonAmpliSeqTranscriptome and The resultsofGeneSetEnrichmentanalysisforgenesdifferentiallyexpressedinUSP8-mutatedandUSP8wild-type MAP USP8 K4 wt tumours( ) aswellthegenesrelated to GABA CCND2 others M Bujko,PKoberand , GABA Breceptoractivation_Homosapiens_R-HSA-977444 Activation of GABAB receptors_Homo sapiens_R-HSA-991365 Cellular Senescence_Homosapiens_R-HSA-2559583 PRC2 methylateshistonesandDNA_Homo Meiotic recombination_Homosapiens_R-HSA-912446 SIRT1 negativelyregulatesrRNAExpression_Homo Activated PKN1stimulatestranscriptionofAR(androgen DNA Damage/TelomereStressInducedSenescence_Homo Condensation ofProphaseChromosomes_Homosapiens_R- DNA methylation_Homosapiens_R-HSA-5334118 Packaging OfTelomereEnds_Homosapiens_R-HSA-171306 RNA PolymeraseIPromoterOpening_Homo Wnt signallingpathway_Homosapiens_P00057 Cadherin signallingpathway_Homosapiens_P00012 Extracellular matrixorganization(GO:0030198) Negative regulationofepithelialcellproliferation Nucleosome organization(GO:0034728) Chemical synaptictransmission(GO:0007268) Cell-cell adhesionviaplasma-membrane Nervous systemdevelopment(GO:0007399) Chromatin assembly(GO:0031497) Anterograde trans-synapticsignalling(GO:0098916) Nucleosome assembly(GO:0006334) Synapse assembly(GO:0007416) Term CDK6 sapiens_R-HSA-212300 sapiens_R-HSA-427359 sapiens_R-HSA-5625886 receptor) regulatedgenesKLK2andKLK3_Homo sapiens_R-HSA-2559586 HSA-2299718 sapiens_R-HSA-73728 (GO:0050680) molecules (GO:0098742) USP8 , CDKN1B , EGFR , , USP8 predominantlyin decreased expressionwas observed functioning adenomas as compared to CD; however, were higherinsampleswith in Higher levelsof Fig.3). found betweenthesetumours(Supplementary difference in theexpression level of expression than as differentiallyexpressedinSCAandCDpatients). signalling ( corticotrophinomas Transcriptomic profilingin USP8 USP8 POMC wt tumours. wt tumours, mut tumoursrevealedslightlyhigher expression was found lowered in non- KCNJ5 CCND2 , USP8 GABBR2 CDC25A , wt tumours( CDK6 Downloaded fromBioscientifica.com at09/25/202112:53:01AM , USP8 and GNAL and mutation. MAPK4 CDKN1B and Overlap 181 EGFR Fig. 2A 10/30 20/278 17/150 18/230 10/80 22/290 15/143 31/456 10/66 21/241 10/61 16/64 19/161 10/40 12/54 10/35 10/34 13/59 11/41 10/32 10/31 9/67 3/39 3/39 :6 GABARD expressionlevel and were observed wereobserved ), whileno Adjusted USP8 < < < < < < < < < < < ) (found 0.0003 0.0003 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0088 0.0002 0.0453 0.0419 0.0381 0.0208 0.0096 0.0096 0.0096 0.0092 0.0092 0.0001 POMC 620 was via freeaccess P

European Journal of Endocrinology patients ( levels of than inpatientswithCushing’s disease, whereasmRNA corticotroph tumourswithregardto with The resultsofgeneexpressionmeasurementswithqRT-PCR. (A)Comparisonofgeneexpressionlevelsincorticotrophtumours Figure 2 Clinical Study The expressionof USP8 B A mutationandwild-typecorticotrophtumours.(B)Comparison ofgeneexpressionlevelsinSCAandfunctioning GABBR2 Fig. 2B ), inlinewiththeresultsof NGS-based , GNAL KCNJ5 and washigherinSCApatients GABRD others M Bujko,PKoberand USP8 werehigherinCD mutationstatus.(C)Spearmancorrelationbetween levels. Thisanalysisshoweda negativecorrelationbetween examined thecorrelationbetween thesegenesexpression GNAL transcriptomic profiling. Since corticotrophinomas Transcriptomic profilingin encode proteins involved in the same pathway, we C Downloaded fromBioscientifica.com at09/25/202112:53:01AM GABBR2 and https://eje.bioscientifica.com KCNJ5 KCNJ5 181 :6 , expressionlevel. GABBR2 and 621 via freeaccess European Journal of Endocrinology https://eje.bioscientifica.com and functioningcorticotrophtumours. Afullcolourversionofthisfigureisavailableat silent corticotrophtumours.(B) Theresultsofimmunohistochemicalstainingevaluation.Comparison ofH-scorevaluesinsilent The resultsofimmunohistochemical KCNJ5detectionintissuesamples.(A)Theexamplesof immunostaininginfunctioningand Figure 3 (Spearman was observed and expressionlevelof ( H-core 146and82.2inSCACDpatients,respectively score as compared to functioning tumours with mean SCA samplesshowedhigherimmunostainingreactivity and functioningcorticotrophinomaswasperformed. was calculated for all 22 patients and comparison of silent examples oftissuestainingarepresentedin samples revealedonlycytoplasmicimmunoreactivity. The did notreachsignificancethresholdlevel).Ninetissue 4/11 adenomascausingCD(differencesintheproportions in7/11SCAsamplesand immunoreactivity wasobserved 11 CDtumours). Both membrane and cytoplasmic IHC wasperformedon22FFPEsamples(11SCAand protein expression Immunohistochemical evaluationofKCNJ5 shown in KCNJ5 P =0.0104; Clinical Study BC A Patient C10 100 µm Patient S5 100 µm and Silent Corticotroph Functioning Corticotroph Fig. 2C Fig. 3 GABBR2 . ). PositivecorrelationbetweenH-score (Spearman R KCNJ5 =0.447; T umors measuredwithqRT-PCR others M Bujko,PKoberand R = P Tu =0.0368). − 0.37; mors Fig. 3 100 µm Patient S2 P Patient C15 100 µm .05 as =0.0095) . H-score . H-score criteria, thebiologicaldifferencesbetweenSCAsand the casestomeetcriteriaforSCAs.Regardlessofdiagnostic exclude misdiagnosisofthesepatients,weconfirmedall included. Aftercarefulverificationofclinicaldatato smaller numberofSCAsamples(11and2samples)were in non-functioningcorticotrophtumours,however, a published reports( found were alsoidentifiedpreviously( deletion inhotspotregionexon14.Allthealterations were singleaminoacidsubstitutionsorcodon ( mutations frequencies were also reported in other cohorts tumours ( the mutationswereidentifiedinabout30–40%of in ourstudyissimilartopreviouslyreportedresultswhere The frequencyof Discussion functioning corticotroph tumours are still unclear ( corticotrophinomas Transcriptomic profilingin 4 , 25 Interestingly, topreviousstudieswealso incontrary , 26 USP8 ). Inourstudy, alltheidentified 3 , mutationsinSCApatients(4/20).Inthe 5 , 23 USP8 https​://doi.org/10.1530/EJE-19-0194 , 24 5 ,

KCNJ5 H-score Patient C2 ). However, bothlowerandhigher 100 µm mutations in CD patients observed mutationsinCDpatientsobserved 100 150 200 250 26 50 Downloaded fromBioscientifica.com at09/25/202112:53:01AM 0 ) no mutations were observed ) nomutationswereobserved 4

SC , A 5 , P= 23 181 0.0104 , :6 24 USP8 , 27 CD ). mutations ​. 622 1 via freeaccess ). European Journal of Endocrinology high expressionof level of in thesetumours.In USP8 (encoding cyclin D2) and As alsofurtherconfirmedbyqPCRevaluation, genes including processes inGSEanalysisduetodifferentialexpression of cell proliferationwasidentifiedamongtheenriched GO of cellproliferation.Negativeregulationepithelial known cancer-related genes involved in the regulation this areourfindingsofidentificationDEGsamong in developmentofcorticotrophtumours( carcinogenesis ( of histone-encodinggenes’expressionwasdescribedin disturbances invariouscellularprocesses.Deregulation organization andfunctionalstatus( in thecompositionofhistonesbyaffectingchromatin the expressionofthisclassgenescausesimbalance of NGSresultsaredifferentiallyexpressed.Alterationin genes (29/49)thatpassedthefiltrationinanalysis USP8 histone variants among DEGs (with lower expression in representation ofgenesencodingreplication-dependent analysis withreactomedatabasewerefoundduetohigh and the high representation of protocadherin-encoding genes pathways wereidentifiedassignificantlyenricheddueto were foundtobeaffectedby cell-cell adhesion,cadherinsignallingandWnt molecular processesandpathways.Processesrelatedto expression ofnumerousgenesinvolvedinmanydifferent corticotrophinomas). Presence of the mutations affects the samples fromfunctionalgroups(silentandfunctioning these samples are more similar to each other than represent aseparatecluster. Intermsofgenesexpression, transcriptomic datashowedthat functional status.Hierarchical clusteringbasedonwhole affect molecularprofileoftumoursregardless adenomas clearly showed that investigated duetoalownumberofpatients. higher profile ofgeneexpressionthan suggest that vice versawasalsoreported( lineage andtheevidenceofSCAtoCDconversion cell Both tumour types develop fromthesame pituitary Clinical Study Cell cycleregulatorshavebeenrecognizedasinvolved A largenumberofdifferentpathwaysinGSE The gene expression profiling of corticotroph FZD1 mut patients).Morethanhalfofallhistone-encoding wt tumours,suggestingincreased cyclinDsignalling POMC CDKN1B inDEGs. USP8 expression.However, thisissuewasnot 29 CDK6 mut SCAsmayhavedifferentmolecular ). (encodingp27/Kpi1inhibitor) and CDC25A USP8 , TGFB2 CDK6 mut tumours we observed low mut tumoursweobserved , aknowncellcycle-related USP8 , expression was higher in 8 USP8 SOX2 others M Bujko,PKoberand USP8 , mutationstatus.These 28 29 mutationsstrongly ). Ourresultsmay , wt SCAs,including USP8 ). Thismayleadto SOX9 30 mut samples ). Inlinewith , and CCND2 SNAI2 .

clearly shown their proteasomaldegradation.Thismechanismwas to deubiquitination of itssubstrates and preventing a consequence,theactivityofUSP8isenhancedleading development such as MET1 ( that thelevelofotherproteinsassociatedwithtumour USP8 sustained EGFRsignallingincorticotrophtumourswith interactions between USP8 and 14-3-3 proteins ( involved in binding 14-3-3 proteins and thus impair mTOR signalling( increased cellproliferationthroughstimulationofAKT/ in humancancers. Its overexpressionresultsinthe atypical MAPkinasethatwasfoundtobeoverexpressed ERK4) with higher expression in patients wasreported recently ( protein level in mutatedandwild-typecorticotrophinoma oncogene ( ( involved incellsignalling( > in termsofgenesexpression withonly66DEGs(withFC active and silent corticotrophinomas differ onlyslightly Unexpectedly, ourresultsshowthatthefunctionally the causeofsilentnatureSCAsremainselusive. few studies. Intriguingly,have been investigated in very functioning andnon-functioningcorticotrophtumours tumours causing CD. Thebiologicaldifferences between expression profilesofSCAsandfunctioningcorticotroph in expression causedbyenhancedactivityof which isconcordantwiththereportedincreaseinACTH 24 in GSEanalysis. EGFR-related pathwaysassignificantlyenrichedforDEGs as also reported previously ( differencein observe comparing manifested byderegulationofmanyproteins.When biological effectnotlimitedtoEGFRpathwaythatitis tumours suggest that the mutation exerts a pleiotropic pathways incomparisonof reported. The large number of DEGsand the enriched (SMO) ( in preventingdegradationofVEGFR2( role of USP8 sustained with this mechanism. Regulatory cell adhesion ( corticotrophinomas Transcriptomic profilingin KCNN4 2.5) found inthis comparison. The identified genesare ) USP8 Mutations in According toourstudyandpreviousresearch ( We have paid special attention to comparison of gene USP8 mutations( mut tumours( 37 , KCNJ5 mut tumoursshowhigher ), frizzled(FZD)( 31 USP8 ). Correspondingly, adifferenceinp27/Kip1 GJA1 in vitro ). We did not find significant differences in mut and 3 32 USP8 , , ). 4 CDH6 ). However, thereisalsoevidence providingtheexplanationof 3 USP8 ). hotspotoccurintheregion Downloaded fromBioscientifica.com at09/25/202112:53:01AM ) or ion channels functioning USP8 and 38 GLI2 USP8 4 , 34 ) andNrdp1( 24 https://eje.bioscientifica.com wt patientswedidnot ) and Cdt2 ( , EGFR -mutated andwild-type 27 ). We also did not find DLC1 USP8 ). 181 expressionlevels, POMC MAPK4 :6 36 , mut tumours is POMC TBX2 ), smoothened 39 expression, 35 (encoding , ) wasalso promoter ) may be RASSF6 33 623 ). As via freeaccess 4 ), , European Journal of Endocrinology https://eje.bioscientifica.com receptor ( in corticotrophAtT-20 cells,stimulationofsomatostatin effectors ofG-protein-coupled receptors( hyperaldosteronism ( calcium mobilization,increaseddepolarization,and result inlossofselectivityforK+,causingNa+influx, familial hyperaldosteronismtype3. adenomas, whilegermlinemutationsofthisgenecause KCNJ5 play apivotalroleinaldosteronesecretion.Somatic on GtEX database). In adrenal gland KIR3.4 seems to highest expressionofallKIR3-encodinggenes(based involved inanegativeregulation ofACTHsecretion, in activationof KIR channels.Since KIR channels are on GtEXdatabase) ( (based mostly expressed in adrenal gland and pituitary and pituitary, withexception of KIR3.4 ( KIR3-encoding genesaremostlyexpressedinbrain potential uponCRHstimulation( cells andtheirinhibitioncontributestofiringactivation shown tobeinvolvedinmaintainingRMPcorticotroph secretion incorticotrophcells( and increaseinCa2+concentrationthattriggersACTH Blocking K+channelsresultsinmembranedepolarization potential (RMP)andpreventmembranehyperpolarization. channels areinvolvedinstabilizationofrestingmembrane activated inwardrectifierpotassiumchannel.KIR3family It encodesKIR3.4(GIRK4)protein,whichisaG-protein- change ofexpressionbetweenSCAsandCDadenomas. expressed. GNAL involved inregulationofmembranepotential( channels ( and functioningtumours.Indeed,genesencodingion of cellsareaspecialinterestwhencomparingSCA Therefore, the genes involved in electrical excitability channels and theinfluxofthese ionsintothe cell( depolarization associatedwiththeopeningofCa activity ofcorticotrophcellsisprovokedbymembrane regulated bychangesinmembranepotential.Secretory ( expressed inSCAsandfunctioningcorticotrophinomas ( cells to differentiationofpituitary CRHR1 prohormone convertase 1/3 gene ( the expression of genes related to ACTH release including 7 TPIT , Clinical Study 12 Four KIR3channelshavebeenidentifiedinhumans. KCNJ5 cells isThe physiological activity of pituitary ) thathavebeenreportedpreviouslyasdifferentially ). , mutationsarefoundinaldosterone-producing , GJA1 AVPR1B 46 KCNJ5 wasidentifiedasaDEGwiththehighestfold , –connexin43)werefounddifferentially 47 ( ) and dopamine receptors ( , V1BR KCNN4 44 43 ) , ). KIR3channelsaredownstream

). In pituitary, HSD11B2 , GABRD others M Bujko,PKoberand 41 9 42 ( ), aswellothergenes , NEUROD1 7 ). KIRchannelswere 10 ). , 12 , KCNJ5 12 ) orgenesrelated 45 KCNJ5 ), KCNJ5 ). As observed ). Asobserved SSTR1 and mutations 48 hasthe GABBR2 ) results ) that is , TBX19 SSTR2 40 2+ ). ). , ,

subunits ofGABAreceptorsB( thehigherexpressionofgenesencoding we observed ( effectonotherones corticotrophs, whileithasinhibitory stimulates some functional subtypes of the cells including GABA actsinaparacrineandautocrinemanner, andit related toGABAsignalling– tumours showedalsohigherexpressionofanothergene in functioningtumours.Additionally, CD-causing well as pathway includesthreeDEGs: pathway foundinReactomedatabase.This activation ofGABAreceptorB(GABARB)wastheonly corticotrophinomas, theenrichmentinprocessesof differentially expressedbetweenSCAsandfunctioning functionally relatedtoinactivenatureofthesetumours. high expressionof mutations occurincorticotroph adenomascausingCDas and the inversecorrelationofexpression observed for GABABreceptor( adult neurons,whereKIRchannelsareoneoftheeffectors of GABAsignallinghasbeenmostextensivelystudied in as effectorsforotherG-protein-coupledreceptors.Therole KCNJ5 stimulation onACTHrelease,itisfairlyprobablethat considering thereportsofnoinfluenceGABARB and subunit incorticotrophcellsareavailable. gonadotrophs ( activityofpituitary specific agonistenhancessecretory function. Itwasshownthatstimulationof overexpression ofonesubunitonlywouldaffectreceptor in functioningadenomasthanSCAs.Itisnotclearhow encoding subunit types ( gated ionchannelconsistingoffivesubunitsdifferent hand, GABARA receptor is a large pentameric ligand- muscimol, andnotwithbaclofen( GABARB, as the cells release ACTH upon stimulation with by stimulatingGABAreceptorA(GABARA)ratherthan 52 in functioningcorticotrophtumoursthanSCAs.The receptor doesnotplayaroleinACTHrelease( cells ( unclear. GABARBsubunitsareexpressedincorticotroph possible functionalconsequencesoftheseresultsare corticotrophinomas Transcriptomic profilingin 49 ). In corticotroph cells, hormone secretion is mediated , KCNJ5 When GSEmethodwasappliedfortheanalysisofgenes In summary, our results show that According toGSEanalysiswithReactome, GNAL 50 50 and α , GABBR2 1-6, , 51 51 playaroleinGABARBsignalling.However, thatsuggesttheoppositerole ofthesegenes. ). AccordinglytothisgeneralroleofGABA, GNAL β ), buttheavailabledataindicatethatthis 1-3, 53 and (encodingG-proteinsubunitalpha)act ), butnodataregardingtheroleofthis γ δ 1-3, ( KCNJ5 GABRD GNAL 54 δ Downloaded fromBioscientifica.com at09/25/202112:53:01AM , ). Incorticotrophtumours we inSCAscanbeconsideredas ε , bothwithhigherexpression , ) isexpressedathigherlevel GABRD π and KCNJ5 GABBR2 46 θ . In anterior pituitary . Inanteriorpituitary ) andonlythegene (describedabove)as 181 , 48 ) andA( :6 ). Ontheother USP8 δ subunitwith hot spot GABBR2 GABRD 49 KCNJ5 624 , 51 via freeaccess ) ,

European Journal of Endocrinology References the manuscript. proofreading for Kiepurski A and Kowalewska M thank authors The Acknowledgements grant SN/GW09/2017. Supported by Maria Sklodowska-Curie Institute – Oncology Center internal Funding be could that interest of conflict perceived asprejudicingtheimpartialityofthisstudy. no is there that declare authors The Declaration ofinterest E​JE-19-0194 at paper the of version online the to linked is This Supplementary data tumours causingCD. in GABAsignallingmayfavourACTHhypersecretion in SCA,whilehighexpressionlevelsofgenesinvolved be involvedinloweringelectricalexcitabilityofthecells Our findings suggest that high found todistinguishSCAsandCD-causingtumours. Expression levelsofrelativelylownumbergeneswere numerous genes involved in different molecular processes. have apleiotropiceffectaffectingtheexpressionlevelsof determine themolecularprofileoftumour. They well as in SCAs albeit with low frequency. 6 5 4 3 2 1 Clinical Study Gabalec F, Beranek M, Netuka D,Masopust V, Nahlovsky J,Cesak T, Perez-Rivas LG, Theodoropoulou M,Ferraù F, Nusser C,Kawaguchi K, Ma ZY, Song ZJ, Chen JH,Wang YF, Li SQ,Zhou LF, Mao Y, Li YM, Reincke M, Sbiera S,Hayakawa A,Theodoropoulou M,Osswald A, Albani A, Theodoropoulou M&Reincke M.GeneticsofCushing’s Ben-Shlomo A &Cooper O.Silentcorticotrophadenomas. 0316-1) Pituitary adenomas. pathological typesofclinicallynon-functioning pituitary Marek J &Cap J.Dopamine2receptor expressioninvarious jc.2015-1453) and Metabolism adenomas causingCushing’s disease. The geneoftheubiquitin-specificprotease8isfrequentlymutatedin Stratakis CA, Faucz FR,Wildemberg LE, Assié G,Beschorner R org/10.1038/cr.2015.20) in Cushing’s disease. Hu RG, Zhang ZY ng.3166) disease. et al Beuschlein F, Meitinger T, Mizuno-Yamasaki E, Kawaguchi K, Saeki Y org/10.1111/cen.13457) disease. 2018 . MutationsinthedeubiquitinasegeneUSP8causeCushing’s 21 ​. 2012 Nature Genetics Clinical Endocrinology 1–9.(doi:10.1007/s11102-018-0864-8) 15 2015 222–226. et al 100 Cell Research . Recurrentgain-of-functionUSP8mutations 2015 E997–E1004. (https://doi.org/10.1007/s11102-011- 47 2018 31–38. 2015 88 Journal ofClinicalEndocrinology Journal others M Bujko,PKoberand 3–12. KCNJ5 (https://doi.org/10.1210/ (https://doi.org/10.1038/ 25 306–317. (https://doi. https​://doi.org/10.1530/ expression may USP8 (https://doi. mutations Pituitary et al .

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52 Clinical Study Pitman KA, Borgland SL,MacLeod B&Puil E.Isovalinedoesnot pone.0118497) PLoS ONE expressing AtT-20 cellsandculturedrathippocampalneurons. activate GABA(B)receptor-coupledpotassiumcurrentsin Endocrinology glandinvitro. stimulating hormonefromtheratpituitary luteinizing hormone,adrenocorticotrophichormoneandthyroid- 2015 1986 10 e0118497. 108 1–8. (https://doi.org/10.1677/joe.0.1080001) (https://doi.org/10.1371/journal. others M Bujko,PKoberand Journal of Journal Accepted 2October2019 Revised versionreceived5September2019 Received 14March2019

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