178

European Journal of Endocrinology 10.1530/EJE-17-0702 ( effort, specificgeneticmutations arerarelyfoundinNFPAs adenomas (NFPAs) remainselusive.Regardless ofintensive The pathogenesisofsporadic non-functioningpituitary HOOK1, CNOT6L P the fastgroup, Results human adenomacellswasevaluated. and pathwayanalyses.Theeffect ofsilencing cummeRbund pipeline.40geneswereselectedforRT-qPCR validationin20GAsbasedonsignificance,fold-change by thetumorvolumedoublingtime(TVDT)median(27.75 months).Datawereanalyzedtophat2,cufflinks and Design andmethods transition (EMT). biomarkers fortumorgrowthpotentialcouldbeidentified,focusingonthespecificroleofepithelial-mesenchymal necessary. We hypothesizedthatfast-andslow-growingGAspresentdifferent geneexpressionprofilesandreliable lacking. Asthegrowthoftumorremnantsishighlyvariable,molecularmarkersforpotentialprediction are Objective Abstract Research andStudyCentre(LV BMC),Riga,Latvia University Hospital,Oslo,Norway, Odense, Denmark, University Hospital,Oslo,Norway, 1 Nicoleta Cristina Olarescu Tove Lekva Camilla Maria Falch pituitary adenomas slow-growing non-functioninggonadotroph expression profiling offast-and Introduction therapy. aggressiveness, theothergenesmightrepresentmarkers fortumorgrowthpotentialandpossibletargetsdrug have higherexpressioninfast-growingtumors.Inaddition to Conclusions with EMTmarkers. Section ofSpecializedEndocrinology, DepartmentofEndocrinology, 1

https://doi.org/10.1530/EJE-17-0702 www.ej < ). There are no reliable molecular markers associated with ). Therearenoreliablemolecular markersassociatedwith Clinical Study

0.05). Thesewere e-online.org : 350genesweresignificantlydifferentially expressed(282genesupregulatedand68downregulatedin : Reliablebiomarkersassociatedwithaggressivenessofnon-functioninggonadotrophadenomas(GAs)are : Fast-andslow-growingGAspresentdifferent geneexpressionprofiles,andgenesrelatedtoEMT 2 , Ivars Silamikelis 4 University ofSouthernDenmark,Odense, P and -adjusted : EightGAsselectedforRNAsequencingwereequallydividedintofast-andslow-growinggroup 1 PCDH18, UNC5D,EMCN,MYO1B,GPM6A PRKACB , 2 , 3 , 4 PROOF ONLY , Arvind Y M Sundaram 6 3 < 3 Faculty ofMedicine,UniversityOslo,Norway, and Department ofEndocrinologyandMetabolism,OdenseUniversityHospital, 1 0.05). Among40selectedgenes,11showedassociationswithTVDT( , 2 © ). 2018EuropeanSociety ofEndocrinology 7 MTDH , Alexander Kirkeby Eieland C MFalch , butnot et al. Printed inGreatBritain MTDH EMCN 5 , Kristin Astrid (metadherin)and , demonstratedinvolvementincellmigrationandassociation 2 Research InstituteforInternalMedicine,Oslo 5 Department ofMedicalGenetics,Oslo in termsofmedicalresources andsocietalcosts. is necessary,observation constituting a significant burden behavior ademandingchallenge. Consequently, prolonged aggressiveness orrecurrence,making thepredictionoftumor gonadotroph NFPAs Fast- andslow-growing 1 andsixEMT-related ( MTDH Published byBioscientifica Ltd. , Marianne Andersen Ø , identifiedasanimportantcontributorto ystese EMCN 7 Latvian Biomedical 1 (endomucin)on , 6 , Kjersti Ringvoll Normann 3 Downloaded fromBioscientifica.com at10/02/202112:15:28PM , Jens Bollerslev SPAG9, SKIL,MTDH, in vitro (2018) Endocrinology European Journal of [email protected] Email to CMFalch should beaddressed Correspondence − 0.669 178 178

migrationof :3 , 295–307 < 1 , R 6 and

< 1 − , 2 0.46, ,6 295 , –307 via freeaccess European Journal of Endocrinology www.eje-online.org focusing onthespecificrole ofEMT. predicting growthpotential oftheresidualtumortissue, postoperative TVDT, andto find reliable biomarkers in fast-andslow-growing tumors, estimatedbyearly expression profiles. We aimedtoidentifytarget genes initial growthvelocities(highvslow)havedifferentgene ( size, invasivenessandpoorresponsetomedicaltreatment content inthesetumorshasbeenassociatedwithtumor the progressionoftumors.ThereductioninE-cadherin and ACTH-producing adenomas have linked EMT to is ahallmarkofEMT( ( with increased motility, invasiveness and even distant changes toshowmesenchymalcellcharacteristics adenomas( pituitary cancers, whichalsoplaysaroleinthepathogenesisof programmingprocessinvarious studied regulatory ( follow-up intervals case’ scenarioforfuturegrowthcanbeatooltotailorsafe the calculationofearlyTVDTasaprediction‘worst- risk ofregrowth( indicatesahigh tumor remnantsfollowinginitialsurgery or lineargrowthmodel( describe the growth of NFPAs by an exponential, logistic and individualizefollow-up( clinical knowledgeoftumorbehaviorandhelptoadjust data abouttumorbiology, wouldsubstantially enhance the adenoma growthkineticsandTVDT, collaboratedwith course oftumorsinseveralcancertypes.Information follow-up investigations and is used to predict the clinical between enabling the clinician to estimate a safe interval presuming thetumorhasanexponentialgrowthcurve, takes foratumortodoubleinvolume.Itiscalculated describes thegrowthvelocitybymeasuringtimeit adenomas ( ( characteristics (i.e.comparinginvasivevsnon-invasive expression according to tumorinvasiveness or recurrence ( and therebyadequatetreatmentofawiderangecancers signaling pathwaysforcorrecthistologicalstratification molecular markers,aswelltodetermineessential analyses haveshowntobeofimportanceinidentifying subgroups ofNFPAs ( common (75%)ofalltheimmunohistochemical 4 3 15 , ). Recentstudieshaveinvestigatedthedifferentialgene Clinical Study , In thisstudy, wehypothesized that GAswithdifferent Epithelial-mesenchymal transition(EMT)isawell- The silentgonadotrophNFPAs (GAs)arethemost 5 17 ) and early recurrent vs non-recurrent pituitary ) andearlyrecurrentvsnon-recurrentpituitary , 18 , 14 19 6 )). Tumor volume doubling time(TVDT) , , 20 15 12 ). , ), andwehaverecentlypresentedthat 16 13 14 ). LossofmembranousE-cadherin ). , 14 2 15 7 ). Geneexpressionprofiling , ). Studiesofgrowthhormone , 8 16 , 7 9 ). Previousstudiesusually ). Epithelialcellsundergo , C MFalch 10 , 11 ). Thepresenceof et al.

ice andsnapfrozeninliquidnitrogen,laterstoredat operation. Thetumortissuewasimmediatelyplacedon 2002 and2009,allsampleswerefromtheprimary atOsloUniversity Hospital between surgery underwent linear) wereestimatedforallsamples( early TVDT and growth models (logistic, exponential or (IHC).Tumor(SF)-1 onimmunohistochemistry volume, luteinizing hormone(LH))and/orsteroidogenicfactor hormones (follicle-stimulating hormone (FSH)and/or stored tumortissueandthepositivityforgonadotroph theavailabilityof not interruptedbynewinterventions), imaging (MRI)examinationsorfiveyearsoffollow-up, regrowth andfourormoreconsecutivemagneticresonance possibility toassessgrowthmodeling(onlytumorswith from alargercohortofpatients( The patientsincluded in this study ( Patients andsamples Subjects andmethods of cDNA. measurements wereperformed withthesamebatch reverse transcriptionpolymerase chainreaction(RT-qPCR) the cDNAwasdiluted1:10 and allreal-timequantitative NJ, USA)using1 Gradient ThermalCylinder(LabnetInternational,Edison, perform reverse transcription in aLabnetMultiGene Transcription Kit(Applied Biosystems)wasusedto an adequate quality. Ahigh-capacity cDNA Reverse samples hadRNAintegritynumbers(RIN) (Nanodrop Technologies, Wilmington, DE,USA).All OD readingsonaNanoDropND-1000Spectrophotometer Technologies) andtheconcentrationswere measuredby was determinedbyAgilent2100Bioanalyzer(Agilent digestion stepforremovalofgenomicDNA.RNAintegrity to themanufacturer’s instructions,includingtheDNase with theQIAGENmiRNeasyMiniKit(Qiagen)according extracted using TRIzol(Invitrogen) and laterpurified After homogenizationoftumortissue,totalRNAwas RNA isolationandreverse transcription the study did not influence treatment or patient follow-up. information aboutthepatient’s genotypewasgiven,and were exclusivelyconnectedtotumorbiology. Thus,no and hospital authority. Thegenetic expression analyses committee (REK no: 2014/635 and REK no: 2014/1680) patients. Thestudywasapprovedbytheregionalethics − gonadotroph NFPAs Fast- andslow-growing 80°C orembeddedinTissue-Tek. Written informed consent was obtained from all μ g RNAinreaction.Afterthereaction, Downloaded fromBioscientifica.com at10/02/202112:15:28PM n 178 n

= 0 wr selected were =20) 88) basedonthe 13 :3 ). Thepatients > 7, indicating 296 via freeaccess

European Journal of Endocrinology selected genes,RT-qPCR wasperformedintheABI To verifythe expressionofthetranscripts RT-qPCR validation ( Pearson correlationwithcompletelinkageinTM4MeV expression. Hierarchical clusteringwasperformedusing expression plotswerecreatedforbothgeneandisoform custom scriptswereusedtocreatetablesandgraphs.Gene ( described in ensembleGTFand CummeRbund, v2.14 calculate thedifferentialexpressionofknowngenes alignment. Cuffdiff, v2.2.1 ( the outputwasalsoprovidedasparametersfortophat2 ( aligning thefirstonemillionreadsusingbowtie,v2.2.3 github.io/picard/) CollectInsertSizeMetrics tool after estimated usingpicard,v1.112(http://broadinstitute. sizewas --transcriptome-index’ asparameters.Library fr-firststrand--no-mixed--no-novel-juncs ‘--library-type the transcriptome using Tophat2, v2.0.13 ( against theHumanensembleGRCh38genomeand Transcriptome alignment added asaspike-induringsequencing. reads aligningtoPhiX(RefSeq:NC_001422.1),whichwas ( v0.33 ( quality reads and adaptors were removed using Trimmomatic concatenated foreachsamplebeforefurtheranalysis.Low- Pre-processing andcleanup Data processing preparation. library generate fastqdatabasedontheindexesusedduring processed usingbc2fastqv2.17.1.14todemultiplexand v1.18.66.3 wasusedforbasecallingandfurther performed ontwolanesofHiSeq2500(Illumina).RTA pooled together, and125 by followingmanufacturer’s instructions.Librarieswere prepkit(Illumina,SanDiego,CA,USA) mRNA library unique indexeswerepreparedusingTruSeq stranded Centre, Oslo,Norway. EightRNA-seqlibrarieswith RNA foreachsample( The RNAsequencing(RNA-seq)wasperformedusing2 Library preparationandsequencing RNA sequencing http://sourceforge.net/projects/bbmap http://mev.tm4.org/ 25 23 Clinical Study ). Rpackagewasusedtovisualizeexpressiondata,and ) toHumanensembleGRCh38cDNAsequences,and 21 ) with recommended parameters. BBMap v34.56 ) withrecommendedparameters.BBMapv34.56 ). n

= 8) at the Norwegian Sequencing 8) attheNorwegian

nt pairedendsequencingwas Cleaned datawerealigned Fastq data from two lanes were Fastq datafromtwolaneswere 24 ) pipeline was used to C MFalch ) was used to remove ) wasusedtoremove et al. 22 ) using μ g of the standard curves. AllRT-qPCRof thestandardcurves. experimentswerein and correlationcoefficients were acquired from the slope were carefully selected. Primers’ amplification efficiency could detectthesameisoformsasdetectedbyRNA-seq amplification due to DNA contamination. Primers that the primerstotheirlocationavoidfalse-positive org genomic DNAsequence(downloadedfrom tested theirspecificityusingBLASTanalysis(NCBI).The data supplementary the primerpairs( edu/primerbank/) orsearched theliteraturetofind amplification wasperformedaspreviouslydescribed( (ep corresponding wells by an automated pipetting system Applied Biosystems)andsamplesweredispensedinthe Reaction mix(withPowerSYBRGreenPCRMasterMix, 7900 (AppliedBiosystems)ontheentirecohort( bFGF and20 A. Theculturewasfedtwo timesaweekwith10 50 fetal calf serum, penicillin/streptomycin (50 31331-028) supplementedwith10 cell fractionwascultured in DMEM/F12(GIBCO, cat. no. Flasks (ThermoScientificcat.no.156340).Theattached Falcon), counted and plated at ca. 10 the cellswerefilteredthrough100 adding humanalbuminandremovedbywashing, at37°C.Thepapainwasstoppedby NJ, USA)for3 min Biochemical Corporation,cat.no.LS003118,Lakewood, dispersed withpapain13.2 small pieces.Afterwashing,thepelletwasenzymatically The blood was removed immediatelyand the tumor cut in no. 12-700F, Verviers, Belgium)andtransportedonice. operation roominL-15(Leibovitz)medium(Lonza,cat. operationwascollectedinthe tissue fromtheprimary a previouslydescribedprotocol( cellswereobtainedfromthreepatientsfollowing Primary Cell cultures In vitro genes in NFPAs, and expressed as relative mRNA levels ( previously shown to be some of the most stable reference the geometricmeanof expression wasnormalizedtotheindexobtainedfrom quantified usingthedelta-deltaCt(ΔΔCt)method.The accordance totheMIQEguidelines.Geneexpressionwas gonadotroph NFPAs Fast- andslow-growing µg/mL), 2.5 Motion ) was used to study exon–intron borders by matching ) wasusedtostudyexon–intronbordersbymatching We usedtheprimerbank(https://pga.mgh.harvard. experiments 5070 CB,Hamburg,Germany).RT-qPCR ng/mL TGFa(R&DSystems, cat.no.233-FB µg/mL heparinand1 Supplementary file1 Supplementary given at the end of this article), and given at the end of this article), and Downloaded fromBioscientifica.com at10/02/202112:15:28PM GAPDH and U/mL (Worthington mM HepesBuffer, 1% μ 178 27 × m cellstrainer(BD ALAS1 5 cells/cm B27w/ovitamin ). Briefly, tumor www.eje-online.org :3 , see section on , seesectionon www.ensembl. Ct levels, as Ctlevels,as U/mL and 2 in Nunc n ng/mL 297 =20). 26 26 via freeaccess ). ). European Journal of Endocrinology www.eje-online.org were boiled at 95°C for 5 min. WB was performed with were boiled at 95°C for 5 min. Buffer (cat.no.39000),was added,andthesamples (cat. no. 23225). Pierce Lane Marker Reducing Sample amount wasmeasuredwith Pierce BCAProteinAssay Kit no. 78430),scraped,andtheproteinwascollected.The mixed withHaltProteaseInhibitorCocktail(100 PBS, lysedusing100 Scientific. Thecells,keptonice,werewashedwithcold extraction reagents were from ThermoFisher WB cat. no.11644793001). measured usingCytotoxicityDetectionKit(Sigma-Aldrich (WB) andIHC( silencing wasvalidatedbyRT-qPCR andbyWestern blot 5 was 5 and scramblecontrolwasused.Thesensestrandsequence EMCN siRNA, ThermoFisher Scientific) specific for human experiments. siRNA(AmbionSilencerSelectPre-designed incubated fortwoorthreedaysbeforeperformingthe (ThermoFisher Scientific, cat. no. 13778075) and were transfectedwiththeLipofectamineRNAiMAX After thecellsreached70–80%confluence,they Silencing andRT-qPCR together toperformdataanalysis. normalized and the mean of each patient were pooled were performed with 3–4 technical replicates/patient, the classicalfibroblastappearance.All it becamemorehomogenousandnoneofthecellshad the culturewasheterogeneousjustafterisolation, Lastly, that after a few passages, even if we observed characteristic of epithelial cells ( as aresultfromthedivisionofsinglecells,typical the beginningofculturing,cellsgrewincolonies FCS andpresenceofB27w/ovitaminA.Moreover, in should favor the growth of neural stem cells due to low ( of thefibroblastgenecollagentypeIalpha2chain strong positiveexpressionof and notfibroblasts.AtthemRNAlevel,cellsshowed adenomacells, conditions selectthegrowthofpituitary were between the2nd and 9thpassages.Ourculture untilthecellswerereadyforpassaging.The 10 days and cat.no.239-A),themediawasreplacedevery COL1A2 ′ -GCUGUUUCAACAUUACAAAtt-3 Clinical Study ′ , -GAAUCUCCCAAACAAAUAAtt-3 GAPDH ) ( Supplementary File6 Supplementary (asapositivecontrol, Supplementary file3 Supplementary μ L/well M-PER(cat.no.78501) SF-1 C MFalch Supplementary file 7 Supplementary ). Theculturemedia andlowexpression ′ Supplementary file8 Supplementary for ). Cytotoxicitywas ′ for in vitro et al. EMCN MTDH studies × MTDH . The ) (cat. and ). ). ) , completely healedorupto33 migrationrate,untilthewoundwas on theobserved 3rdto12th was made,aswellevery ocular motic N-WF 10 Images weretakenwithMoticAE200,objectivemotic4 Wound healingassay Tokyo, Japan). analyzed withtheMultiGaugesoftware(Fujifilm, (1:10.000). Proteinlevelswereadjustedto antibody Antibody (CellSignaling)wasusedassecondary Scientific (1:1000)( Polyclonal Antibody(PA5-21395), ThermoFisher antibody ab96315,Abcam(1:500)andEndomucin (1:5000) bothfromCellSignaling,andAnti-Endomucin (1:2000) ( used: Lyric/Metadherin (D5Y8R) XPRabbitmAb#14065 20 months postoperatively. The samples were divided except twosamplesshowed tumorremnantatthree operation, and allmacroadenomas from the primary The samples included in this study ( RT-qPCR analysis Characterization ofpatientsamplesforRNA-seqand Results A used. TheanalyseswereperformedusingSPSS,version21. repetitive measuresmodelwithBonferronicorrectionwas To assess the differences in the wound-healing assay, differences between silenced cells and scramble controls. Student’s sample independent associations.One-wayANOVA andunpaired association analysiswasusedtoquantifybivariate Mann–Whitney Differences in patientdemographics were analyzedby continuous variablesandascountsforcategoricaldata. are presentedasmeans by visualmethods(histogramsandQ-Qplots).Thedata All measurementswerecheckedforanormaldistribution Statistics imagej.net/Fiji/Downloads wound borders)wasmeasuredusingFijiImageJ( expected toloseitsfunction.Cellmigration(µmbetween gonadotroph NFPAs Fast- andslow-growing P μ value g proteinperlane.Thefollowingantibodieswere < MTDH 0.05 wasconsideredsignificant. t test(two-tailed)wereusedtoevaluatethe ) and U test.Anon-parametricunivariate EMCN × ± , mitocam 2300 after the scratch Downloaded fromBioscientifica.com at10/02/202112:15:28PM

s GAPDH . ). Anti-rabbit IgG, HRP-linked ). e . m . orasmediansandIQRfor hours whensilencingwas (14C10)RabbitmAb 178 n :3 hour depending 0 wr GAs were =20) GAPDH https:// 298 and via freeaccess × ,

European Journal of Endocrinology velocity dividedintofast-orslow-growingtumorsbypostoperativeTVDTmedian. *Samples forRNAsequencingareincludedinsamplesvalidationwithRT-qPCR; **InvasivenessmeasuredbyKnosp–Steinercriteria;***Growth Growth velocity***(fast/slow) Preoperative volume(cm to EMT( CD44 ROCK2 UNC5D ADGRB2 were involvedincelladhesion( related pathways( literature search withthemainfocusoncancer-andEMT- slow group;therefore,wealsobasedtheselectionon differentially regulatedpathwaysbetweenthefastand http://biit.cs.ut.ee/gprofiler/ ( < with RT-qPCR basedontheirsignificance ( genes wereselectedforvalidationandinvestigation group (fold-change between 1.9and 18.4) ( and 32.0),68geneswereupregulatedintheslow upregulated inthefastgroup(fold-changebetween1.9 the slowgroup( expressed genes( RNA-seq analysis identified 350significantly differentially RNA-seq samples. or growthmodelbetweensequencedandtheremaining difference in gender, preoperative volume, initial TVDT Steiner score invasive tumors(5vs1patientsshowingaKnosp– were older(68( growing tumors.Thepatientswithsequencedtumors RT-qPCR andRNA-seqstudiesofthefast-slow- a descriptive baseline data of the samples used in both with RT-qPCR ontheentirecohort. genes from the sequencing analysis were validated fast andfourslow)wereselectedforRNA-seq.Selected Eightsamples(four the TVDTmedian(27.75 months). into afast-andslow-growinggroup,respectively, by Growth model(logistic/linear/exponential) Invasive** (yes/no), Tumor volumedoublingtime(TVDT)(months) Age (years) Gender (female/male) Table 1 Supplementary file 4 Supplementary 0.05), fold-change,expressionplotsineachsample Clinical Study , UNC5D and

and , Fig. 1C Clinical characteristicsofpatients. FBLN1 FNBP1L ≥ , ATP1B2 MDK ). 3 ontheleft/rightside).Therewasno , 21 PCDH18 Supplementary file2 Supplementary P n -adjusted Fig. 1B ) vs54(

=

14 and ) andmigration ( ) and pathway analyses (g:Profiler, ), cytoskeleton( 3 ), n and ,

FBLN1 = EMCN 12

). Therewerenosignificantly 14 < 0.05) betweenthefastand ), ( C C MFalch ). Ofthese,manygenes UACA ). 17 genes were related P , CD44

=

0.02) andhadmore ): 282geneswere , LGALS3BP al 1 Table GPM6A HOOK1 , MTDH et al. i. 1A Fig. P , provides , MYO1B -adjusted , MTDH ROCK2 , RT-qPCR PXN 6.02 (4.97) 27.8 (21.6) ). 40 57 (20) 10/5/5 10/10 7/13 6/8 , , , ,

( n

=

20) associations withTVDT( cohort ofGAs. and differentimagisticinvasivenessparametersinthe and initialTVDT, pre-andpost-operativetumorvolume Figure 2A RT-qPCR protein p53)geneexpression (datanotshown). TVDT, was found.Therenoassociation betweentheinitial pre- and post-operative volume no significant association expressed intheinvasivetumors. Betweeninvasivenessand tumors, withtheexceptionofLRCH2thatwashigher in between invasive and non-invasive with postoperativeinvasiveness.Therewasnodifference (positively), and FBLN1 and negativeassociationswith invasiveness for We foundpositiveassociationswiththe preoperative in thelinearandahighexpressionlogisticgrowth. expression intheexponential,anintermediate with growthmodels,meaningthattheyexhibitedalow selected genes ( negatively withpostoperativeinvasiveness.Someofthe P in additiontoapositiveassociationwith ( tumor volumewasnegativelyassociatedwith correlating withage( and genderorage,with was nosignificantassociationbetweenselectedgenes preoperative volumewas volume ( SKIL GPM6A ( gonadotroph NFPAs Fast- andslow-growing R i. 2B Fig.

=

=

0.020). Ofthese,only

− 11 ofthe40genesshowedsignificantnegative Preoperative volume was associated with postoperative Preoperative volumewasassociatedwithpostoperative , MTDH 0.525, , MTDH P3H3 andsixgenesknownasrelatedtoEMT( RNA seq( ). Thesewere R 6.69 (2.41) 29.6 (73.9) showstheassociationsbetweenselectedgenes

= 68 (21)

0.747, P 5/1/2 , and HOOK1 4/4 5/2 4/4

, = EMCN

.2) and 0.025) n i. 2A Fig.

BSG = P3H3 ANXA11 P

8)*

=

and , . Amongthese,only 0.003). The only gene associated with 0.003). Theonlygeneassociatedwith CNOT6L R PCDH18 (negatively), remained associated (negatively),remainedassociated

= ) showed a positive association ) showed apositive association Downloaded fromBioscientifica.com at10/02/202112:15:28PM

− ANXA11 FOS MKI67 , CNOT6L 0.473, LRCH2 5.23 (4.94) 19.7 (10.8) Fast CNOT6L − 60 (37) ( 7/2/1 0.669 10/0 3/5 2/8 and , ( R MDK UNC5D n (Ki-67)and

= = P

as the only exception astheonlyexception 10) 0.609,

( = , ≤ PRKACB KIF5B was also associated wasalsoassociated

, R 0.035). Postoperative Postoperative 0.035). LGALSSBP R 178

= ≥

− www.eje-online.org − , :3 LRCH2 0.633, 0.466, P EMCN

= FOS and

). 0.021). There There 0.021). Slow 5.44 (3.72) 38.6 (55.0) TP53 56 (17) ( 3/3/4 and 0/10 , P , 3/3 5/5 WFDC2 R MYO1B

( P = n

FNBP1L (tumor (tumor = HOOK1

SPAG9

< 0.005), 0.005), =

0.556, 0.556, KIF5B

299 10)

0.05) via freeaccess , , ,

European Journal of Endocrinology www.eje-online.org involvement ofselectedgenes ( fold-change indicateshighergene expressionintheslowgroup.(C)Epithelial-mesenchymal transition(EMT)andcancer non-functioning gonadotroph adenomas.Positivefold-changeindicateshighergeneexpression inthefastgroup.Negative ( of 350differentially expressed genes,282genesupregulatedand68downregulated inthefastgroupwerefound S Heatmap showinggeneexpressionprofilesclusteredacross samples andgenes.Geneexpressionlevelsforeachadenoma( Significantly differentially expressedgenesbetweenfastandslownon-functioninggonadotrophadenomasbyRNA-seq. (A) Figure 1 P = -adjusted Clinical Study slow, F = fast) arepresentedinhorizontalrowswithcolorsindicating upregulated (red)ordownregulated(blue)genes.Atotal < 0.05). Eachcolumnrepresents a singlegene.(B)Distributionoffold-changeforselectedgenes ( n

= 40). Thesymbol‘x’markstheEMT- and cancer-related genesrespectively. C MFalch et al. gonadotroph NFPAs Fast- andslow-growing Downloaded fromBioscientifica.com at10/02/202112:15:28PM 178 n

= :3 40) infastvsslow n =8, 300 via freeaccess European Journal of Endocrinology between initialTVDTandsignificantly associatedgenes( optic chiasm,3 and alsobythetumor’s superior expansion(0 in thenon-functioninggonadotroph adenomas( TVDT, growthmodels(exponential, linearandlogistic),pre-post-operativevolume, pre-andpost-operativeinvasiveness RT-qPCR validationof40selectedgenes.(A)Associationsbetweenrelativegeneexpressionthegenes( Figure 2 Clinical Study = lifting theopticchiasm,4 C MFalch = blockage of interventricular foramina). (B) Scatter plots representing the associations blockage ofinterventricularforamina). (B)Scatterplotsrepresentingtheassociations = et al. no superiorgrowth,1 n

= 20). Invasivenesswasmeasured accordingtotheKnosp–Steinercriteria( n

= 11) inthenon-functioninggonadotroph adenomas( gonadotroph NFPAs Fast- andslow-growing = upward convexbulgingofthe sella roof,2 Downloaded fromBioscientifica.com at10/02/202112:15:28PM 178 www.eje-online.org :3 n = =20). n 40) and initial initial and =40) touching the touching the 45 301 ), ), via freeaccess European Journal of Endocrinology www.eje-online.org technical replicates/patient;* Data arepresentedasmean scramble ctrlvs cells. (CandD) scramble control(scramblectrl) vssilenced vs silenced cells. (A) Silencing of Figure 3 between patients( though therewasagreatdifferenceincells’behavior cells migrated at a slower rate in all three patients, even migration The wound-healingassaywasperformedtoassesscell Migration assay after twoandthreedays( difference betweensilencedandscramblecontrolcells for EMCN.LDHcytotoxicityassayshowednosignificant were unable to produce data on WB protein expression Due tothedifficultieswithspecificantibodies,we in by 68% was observed a significantreductionin D in protein expression by WB (18%, controls (81%, was significantlyreducedin aggressive phenotype). being associatedwithalowerTVDT(i.e.more negative associationswithTVDT, higherexpression in vitro We chosetwogenes, adenoma cells Silencing ) andIHC( Clinical Study studies.As depicted in MTDH MTDH MTDH MTDH in vitro Supplementary file3 Supplementary mRNAlevelsinscramblecontrol(scramblectrl) MTDH siMTDH P (

< and and siMTDH .0) ( 0.001) Fig. 4A . Itdemonstratedthatthe proteinexpressionbyWestern blotin . EMCN MTDH EMCN GAPDH ) cells.(B) siEMCN MTDH ). Theonlysignificantdifference Supplementary file5 Supplementary ± Fig. 3A EMCN P inprimaryhumanadenoma

s

. ≤ inprimaryhuman and e wasusedasreferencegene. . .5 ** 0.05; m Fig. 2B siMTDH . mRNAgeneexpression ; cells ( ). Asignificantreduction C MFalch n mRNAgeneexpression EMCN EMCN ptet, 3–4 patients, =3 ) wasfound.Similarly, P .0) ( =0.001) P , both genes showed

≤ P cellsvsscramble mRNAlevelsin EMCN .1 *** 0.01; .4) ( =0.046) , formechanistic et al. ( Fig. 3C siEMCN ). P

Fig. 3B siMTDH ≤ 0.001. ) and ).

showed). in geneexpressionofthetwoEMTmarkers(datanot difference between ratio washigher( expression of significantly higher( between CDH1 We investigatedifthegeneexpressionofEMTmarkers E-cadherin andN-cadherin cells ( between controlsand (AUC) and found a significant difference under thecurve 6 in cellmigrationfor endocrine neoplasiaX(MENX) affectedratsbymRNA aggressiveness. Thesecond study performedinmultiple evaluated inregardstomarkers ofinvasivenessand network inthemTORpathway, andthedatawerenot chose tofocusmostlyonthe lncRNA-mRNAco-expression comprising at least five different cell types, the authors subtype (i.e.gonadotrophs)tothenormalpituitary, comparison ofatumorcontainingmainlyonecellular tissues ( RNAs (lncRNA) and mRNAs inGAsvsnormal pituitary study presenteddifferentiallyexpressedlongnon-coding analyzed thesetumorswithsomelimitations.Thefirst literature is sparse in this manner and to date two studies mRNA expressioninfast-vsslow-growingGAs.The regrowth, andapotentialmedicaltarget. a valuablebiomarkerforGAstumoraggressivenessand migration but not involvement inthegrowthvelocity. Furthermore, parameters, suggestinganadditionalrolebehindthe selected genes werealso correlated to classical MRI invasion six ofthemwererelatedtotheEMTprocess.Some 11 showedsignificantassociationswithinitialTVDTand further validationinalargercohortofpatients,butonly the 350differentlyexpressedgenes,40wereselectedfor human GAspresentdifferentgeneexpressionprofiles.Of The presentstudydemonstratesthatfast-andslow-growing Discussion In the gonadotroph NFPAs Fast- andslow-growing h inpatient73( Our study is the first to investigate genome-wide Fig. 4A (E-cadherin)and siMTDH 28 EMCN siMTDH in vitro ). Inadditiontotheconcernregarding and CDH2 , was involved in pituitary adenomacell , wasinvolvedinpituitary cells,thegeneexpressionof , makingitscodedprotein,metadherin, B cellsandscramblecontrols( Fig. 4B ). P siEMCN was observed, butthe wasobserved, P

=

= 0.039). Therewasnosignificant siMTDH siEMCN 0.004), nosignificantreductionin CDH2 Downloaded fromBioscientifica.com at10/02/202112:15:28PM ). Lastly, wecalculatedthearea cellsandscramblecontrols (N-cadherin)wouldchange cells,butnotfor cellswasrecordedafter 178 :3 CDH1/CDH2 CDH1 siEMCN i. 5 Fig. MTDH 302 was via freeaccess ). ,

European Journal of Endocrinology *** when calculatingAUC.Dataarepresentedasmean was recordedafter6 study ( it difficulttodirectlycompare theresultstoour data onthehistologicalsubtype waspresentedmaking biomarkers bymRNAmicroarray inNFPAs, butno two recentstudiesdescribed invasive-relatedcandidate differentially expressedgenesinourstudy. Inaddition, ( of gonadotroph cells both in rats and humans survival regulated bytheSF-1,asapromoterofproliferation/ tumors andnormalpituitary, identified microarrays affymetrix,againcomparinggonadotroph migrating atthesamerateasscramblectrlcells.Theonlysignificantdifference incellmigrationforsilenced siMTDH 15, 24,27and29 and 31 siMTDH Wound-healing assay. (A)Wound-healing assayonsilenced Figure 4 29 Clinical Study ). However, wewereunabletofind P

≤

0.001; AUC,areaunderthecurve.Scalebar, 500 h ( 5 cellsandscramblectrlcells.(B)Wound-healing assayonsilenced cellsmigratingataslowerratethanscramblecontrol(scramblectrl)cells.Asignificantdifference wasobservedafter6 ,

P 30

= 0.03 and0.021)inpatient73(p73),after9,1224 ). h ( P h (

= 0.04, 0.029,0.025,0.019and0.019)inpatient86(p86).AUCshowsasignificantdifference between P

= 0.014) inp73.Nosignificantdifference wasobservedbetween C MFalch CYP11A1 CYP11A1 et al. ± asoneof

s . , agene e µm. . m . ; n MTDH

= 3 patients,4technicalreplicates/patient;*

( h ( genes associatedwithaggressive growthbehavior. designed toidentifygenesinvolved intumorigenesis,but expressed inourstudy, althoughourstudywasnot the somatic mutated genes were found to be differently in theetiologyofsporadic GAs( indicating thatthesegeneswereunlikelytocontribute in thevalidationsetdidnotrevealanymutations,thus mutations. Moreover, DNAsequenceanalysisofthese mutations inindependentgeneswithnorecurrent sequencing insevensporadicGAsidentified24somatic siMTDH gonadotroph NFPAs Fast- andslow-growing P

= The only studyperformingwhole-exome DNA 0.013, 0.008and0.048)inpatient83(p83)after12, EMCN ) cells.Thepictures(cellsarefromp.83)illustrate ( siEMCN ) cells.Thepicturesshow siEMCN Downloaded fromBioscientifica.com at10/02/202112:15:28PM cellsandscramblectrl P

31 ≤ 178 .5 ** 0.05; ). Indeed,noneof EMCN www.eje-online.org :3 siEMCN ( P siEMCN

≤ 0.01; cells ) cells 303 via freeaccess European Journal of Endocrinology www.eje-online.org role. Similarly, astudy inhumanglioblastomacellsshowed a moreepithelialandlessaggressive profile,reassuringits of mesenchymal cells. Moreover, silencing of contributor incellmigration, animportantcharacteristic and thewound-healingassay demonstrated significantly higher expressed in the fast-growing tumors, tumorigenesis ( a majoractorinpituitary that could,intheory, beeasy toapproachifprovedbe in multiplemyeloma( shown thatBortezomibtreatmentcansuppress as anoncogeneisrecognized( MTDH code forproteinspossibletotargetbymedicaltreatment. adenomas. Forthe major component of the tumor growth in gonadotroph selection criteria,wecannotconcludethatEMTisthe corticotroph adenomas( the aggressivenessandprogressionofsomatotroph MKI67 not findanyassociationsbetweengeneexpressionof adenomas ( in pituitary might beusedasmarkersofaggressivenessandrecurrence need for surgery. Despite their limitations, Ki-67 and p53 represents clinical criteria for tumor aggressiveness and the and opticchiasm,ratherthanthelateralinvasiveness, as estimated by TVDT and the threatening of optic nerve beginning ofatumors’invasivecourse.Thegrowthrate these two categories can be challenging, especially in the invasion. However, differentiatingradiologicallybetween and non-invasivetumorsbasedontheircavernoussinus of tumorremnant( estimating earlygrowthvelocitythatcanpredictthecourse asaneffectiveparameterof that initialTVDTmightserve between moreandlessaggressiveGAs,basedonthefact replicates/patient; * are presentedasmean (scramble ctrl)cells. CDH1/CDH2 CDH1 Epithelial-mesenchymal transitiongenes.mRNAlevelsof Figure 5 Clinical Study MTDH EMT isshowntobeofsignificantimportancein We mainlylookedattheinitialTVDTtodistinguish (E-cadherin)and and is extensively studied in other cancers and its role , aknownregulatorofEMT( insilenced TP53 andTVDT, P GAPDH

≤ 13 in vitro

± .5 ** 0.05; CDH2 MTDH ). Earlierstudiescomparedinvasive

s 35 . e 32 . 15 wasusedasreferencegene.Data m ). EMCNisamembraneprotein (N-cadherin)andtheratioof . , ; ( , study, wechosegenesthat n P 33 siMTDH MTDH 17

≤ =

). However, ourstudy did 3 patients,4technical .1 *** 0.01; , 34 C MFalch 18 ). Ithasrecentlybeen and ). However, usingour ) andscramblecontrol P EMCN

≤ 36

et al. 0.001. ). MTDH MTDH . 37 ), was tobea MTDH led to

( adenomacellsandourpremiseswereincorrect, pituitary as: ( EMCN conditions ( and has a critical role under resting and inflammatory that association withEMTmarkers.Ithasrecentlybeenshown cancer treatments( that the integration of multiplesignaling pathways, suggesting prostate, glioma,esophagealandhepatocellular, through progression inmanycancerstypes,includingbreast, Recent studieshaveshownthat these genes( growth ortargetsforpotentialmedicaltherapy. Threeof represent useful markers for aggressiveness and tumor the initialTVDTshouldbefurtherstudied,astheymight is notinvolvedintumoraggressiveness. although thereisanassociationwithearlyTVDT, for adenomaaggressivenessandberesponsible the pituitary adenomacells(e.g.endothelialcells)maydrive pituitary such asitseffectcouldberecorded,( did notallowustoadequatelysilencethe showed overexpression of mesenchymal markers ( and alsowhenupregulating E-cadherin anddecreasedtheexpressionofN-cadherin that knockdownof it shouldberegardedwith cautionsinceotherfactors invasiveness. Althoughthis is aninterestingobservation, they haveanegativeassociation withthepostoperative KIF5B connections inthebrain( role intheestablishmentandfunctionofspecificcell–cell the protocadheringenefamilyandisthoughttoplay a and probablysynapseformation( neuronal plasticityandfilopodiaoutgrowthmotility differentiation andmigrationofneuronalstemcells, and isinvolvedinneuronaldifferentiation,including with TVDT). change onRNA-seq)and characterization are tumors. Two othergenesthatdefinitelyneedfurther as thegeneswerehigherexpressedinfast-growing results somehowcontradicttheirdescribedfunctions, related toEMTsuppression( gonadotroph NFPAs Fast- andslow-growing 2 ) technicalchallenges related to EMCN EMCN We thattumorswithhigh observed The remainingninegenescorrelatingsignificantlyto 1 MTDH EMCN ) weremoreinvasive,butalso easiertoremovesince couldbeexplainedbydifferentmeans,such EMCN expressionatRNA-seqandRT-qPCR levels,( didnotshowinvolvement in cell migration, nor preventsleukocyte-endothelialcelladhesion mayrepresentapotentiallyvaluabletargetin 36 SKIL doesnothavearoleincellmigrationof GPM6A ). Thelackofeffectoncellmigrationby , CNOT6L 34 GPM6A is highly expressed in brain tissue MTDH ). Downloaded fromBioscientifica.com at10/02/202112:15:28PM PCDH18 43 and increasedtheexpressionof (thehighestpositivefoldof ). MTDH 39 HOOK1 , (thehighestassociation MTDH 42 40 ). expressionthecells , 178 PCDH18 3 41 in vitro ) codeforproteins ) othercellsthan promotestumor :3 ). However, our LRCH2 EMCN belongsto culturing EMCN gene 304 and 38 via freeaccess 4 ). )

European Journal of Endocrinology was measured retrospectively in postoperative growing before andafteroperation. Inthisstudy, initialTVDT profile, andsubsequentlythe tumorbehavior, issimilar Fourth, itisnotknownto date,iftheGAsmolecular our data should be performed in other subtypes of NFPAs. Consequently, beforeextrapolating,further validationof different clinicalbehaviorandmolecularprofiles( evidence showsthatthedifferentNFPA subtypeshave of GAsanddidnotincludeothertypesNFPAs. Clear perform thepresentstudyinawell-characterizedcohort cohort ofGAs.Third,wehavedeliberatelychosen to However, the validationstudywasperformedinalarger in vitro a limited number of samples were sequenced, and the compute growth models and initial TVDT ( follow-up. However, to thesecriteria were necessary before thesufficient might haverequired re-intervention follow-up wasobtained,andthefastestgrowingtumors have endedfollow-upbeforefourMRIexamsorafiveyear a selectedsetoftumors.Theslowestgrowingtumorsmight cells. approach and ( characterization ofthecohort,( thorough clinical,imageandimmunohistochemical MTDH assay, humanadenomacells,justonegene(i.e. inprimary when twoofthegenesweretested seq studiesinlargercohortsofpatients.Furthermore, and emphasizingtheneedforvalidationofsmallRNA- initial TVDT, beingindisagreementwithRNA-seqdata of theselectedgenesdidnotshowanyassociationwith were inagreementwiththehypothesis.However, therest would haveresultedinahighernumberofgenesthat perhaps alargercohortofGAsinthevalidationstudy were closetoreachstatisticalsignificance,suggestingthat the RNA-seqdata.Anotherfourthofselectedgenes genes showedassociationswithinitialTVDT, supporting adenomas. Interestingly, justone-fourthofourselected criteria tofurthertestourhypothesisinalargercohortof we carefully selected genes based on various relevant identified genesinthetumorpathology. Accordingly, a broader understanding of the importance of the mechanistic of dataisgenerated,andfurtherinvestigationswith interfered. (debulking vstotalremoval)andpatients’age,mighthave such asperioperativeconditions,theoperativeintention Clinical Study Our studyhaslimitations.First,wemighthavechosen The strengths of thepresentstudy are: ( When performing RNA-seq studies, a large amount ) showedtohaverelevance. studies were performed only in three patients. in vitro 3 ) the use of human primary adenoma ) theuse of human primary studiesareofgreatvaluetogain C MFalch in vitro 2 ) thetranslational , inafunctional et al. 13 ). Second, 1 ) the 44 ). This is linked to the online version of the paper at Supplementary data considered asapotentialdrugtarget. promising biomarkerinfast-growingGAsandmaybe of patients,and with mechanisticstudies. RNA-seq needtobefurthervalidatedinalargercohort different molecularprofiles,butgenesidentifiedby entire cohortofdifferentiallyexpressedgenes. selected ,wedidnotacquireproteindatainthe present study shows or invasive/non-invasiveadenomas.Lastly, althoughthe than bythesimpler, widerusedcriteriaasmacro/micro- approach in characterizing the growth potential of GA, TVDT, evenonpostoperativerecordings,isamoreprecise regarded asalimitation,weconsiderthatmeasuring tumor.potential oftheprimary Althoughthismaybe remnants andmightnotaccuratelyreflectthegrowth References Rikshospitalet, Oslo,Norway, forthetechnicalassistancewithWB. and ResearchInstituteofInternalMedicine,OsloUniversityHospital culture, andtoChristianeFilionMyklebust,DepartmentofHaematology, Oslo UniversityHospital,Oslo,Norwayforthetechnicalassistancewithcell Laboratory forNeurosurgicalResearch,Institute Surgical Research, The authorsthankCecilieSandbergandEmilyPalmero,VilhelmMagnus Acknowledgments of themanuscript. C MFandNOwrotethemanuscript.Allauthorsacceptedsubmission interpretation ofdata.CMFandNOperformedthestatisticalanalysis. database. CMF, NCO,JBandTLperformedtheacquisition,analysis K A out thelaboratoryexperiments.AYMSandIanalyzedRNA-seqdata. M Adesignedtheresearchstudy. CMF, NCO,KRN,TLandAEcarried C MF, NCOandJBformulatedthescientificquestion.M F, NCO,JBand Author contributionstatement Nordisk Foundation(grantnumberNNF16OC0023192,2017)(CMF). The studywassupported in part by ascholarships grant fromTheNovo Funding perceived asprejudicingtheimpartialityofthisstudy. The authorsdeclarethatthereisnoconflictofinterestcouldbe Declaration ofinterest EJE-17-0702. gonadotroph NFPAs Fast- andslow-growing 1

Ø Zhou Y, Zhang X&Klibanski A.Geneticandepigenetic mutations mce.2013.09.006) and CellularEndocrinology adenoma. of tumorsuppressivegenesinsporadic pituitary In conclusion,fast-andslow-growingtumorshave acquiredtheclinicaldataand elaborated the comprehensive clinical in vitro 2014 Downloaded fromBioscientifica.com at10/02/202112:15:28PM 386 mechanistic data for two 16–33. 178 (https://doi.org/10.1016/j. https://doi.org/10.1530/ www.eje-online.org :3 MTDH Molecular 305 isa via freeaccess

European Journal of Endocrinology www.eje-online.org

15 14 13 12 11 10 6 9 8 7 5 4 3 2 Clinical Study Lekva T, Berg JP, Fougner SL,Olstad OK,Ueland T&Bollerslev J. Guarino M, Rubino B&Ballabio G.Theroleofepithelial- Oystese KA, Zucknick M,Casar-Borota O,Ringstad G&Bollerslev J. Chen Y, Wang CD, Su ZP, Chen YX,Cai L,Zhuge QC&Wu ZB. Tanaka Y, Hongo K,Tada T, Sakai K,Kakizawa Y&Kobayashi S. Monsalves E, Larjani S,LoyolaGodoy B,Juraschka K,Carvalho F, Marko NF, Coughlan C&Weil RJ. Towards anintegratedmolecular Hsu CY, Guo WY, Chien CP&Ho DM.MIB-1labelingindex Kucharczyk W,Balogun JA, Monsalves E,Juraschka K,Parvez K, Honegger J, Zimmermann S,Psaras T, Petrick M,Mittelbronn M, Yu SY, Hong LC,Feng J,Wu YT &Zhang YZ.Integrativeproteomics Galland F, Lacroix L,Saulnier P, Dessen P, Meduri G,Bernier M, Hoadley KA, Yau C, Wolf DM, Cherniack AD,Tamborero D, Nishioka H, Inoshita N,Mete O,Asa SL,Hayashi K,Takeshita A, org/10.1210/jc.2012-1760) Endocrinology andMetabolism from alargecohortofpatientswith acromegaly. of epithelialmesenchymaltransition insomatotrophadenomas Gene expressionprofilingidentifies ESRP1asapotentialregulator 305–318. mesenchymal transitionincancerpathology. org/10.1007/s12020-017-1314-5) a tooltotailorsafefollow-up. adenomas; Early postoperativegrowthinnon-functioningpituitary 333–342. a systematicreviewandmeta-analysis. adenomas: ofpostoperative nonfunctioningpituitary Natural history (https://doi.org/10.3171/jns.2003.98.2.0359) age, andMIB-1index. adenomas: correlationsamongtumorvolumedoublingtime,patient Growth patternandrateinresidualnonfunctioningpituitary (https://doi.org/10.1210/jc.2013-3054) ofClinicalEndocrinologyandMetabolism Journal adenomasandhistopathologicalcorrelates. patterns ofpituitary Kucharczyk W, Kulkarni A,Mete O,Gentili F, Ezzat S 2010 adenoma. doubling timeinpituitary correlated withmagneticresonanceimagingdetectedtumorvolume (https://doi.org/10.1007/s12022-014-9347-2) behavioral characteristics. gland: aninstitutionalreviewoftheirclinicalimagingand Mete O, Gentili F&Zadeh G.Nullcelladenomasofthepituitary org/10.1530/EJE-07-0502) ofEndocrinology European Journal adenomasinpatientsreferredforsurgery.functioning pituitary Ernemann U, Reincke M&Dietz K.Growthmodellingofnon- jocn.2012.01.038) Neuroscience adenomas. resected non-functionalpituitary and clinicalstrategytopredictearlyrecurrenceinsurgically 8923–8930. adenomas. of non-functioningpituitary and transcriptomicsidentifynovelinvasive-relatedbiomarkers ERC-10-0018) Endocrine-Related Cancer adenomasbasedonmicroarrayanalysis. non-functioning pituitary Differential geneexpressionprofilesofinvasiveandnon-invasive Gaillard S, Guibourdenche J,Fournier T, Evain-Brion D 929–944. classification withinandacrosstissuesoforigin. al Ng S, Leiserson MD,Niu B,McLellan MD,Uzunangelov V 2015 adenomas. of clinicallynonfunctioningpituitary roleoftranscriptionfactorsintheaccuratediagnosis complementary Fukuhara N, Yamaguchi-Okada M, Takeuchi Y &Yamada S. The . Multiplatformanalysisof12cancertypesrevealsmolecular 162 26 349–355. 1027–1033. (https://doi.org/10.1016/j.cell.2014.06.049) (https://doi.org/10.1080/00313020701329914) (https://doi.org/10.1159/000339823) (https://doi.org/10.1007/s13277-015-4767-2) 2012 (https://doi.org/10.1007/s12022-015-9398-z) 19 1535–1540. (https://doi.org/10.1530/EJE-09-1100) Journal ofNeurosurgery Journal 2010 Endocrine Pathology 2012 17 Endocrine 2008 361–371. (https://doi.org/10.1016/j. 97 European Journal ofEndocrinology European Journal E1506–E1514. C MFalch 158 Neuroendocrinology 2017 Tumor Biology 287–294. (https://doi.org/10.1677/ 2003 2015 57 Pathology 2014 Journal ofClinicalJournal 35–45. Cell Journal ofClinical Journal Endocrine Pathology et al. 98 26 2014 99 (https://doi. (https://doi. et al 359–365. 2016 63–70. 2007 1330–1338. et al (https://doi. . Growth 2012 et 158 . 37 39

96

gonadotroph NFPAs Fast- andslow-growing 27 21 20 19 18 17 16 30 29 28 26 25 24 23 22 Murrell W, Palmero E, Bianco J,Stangeland B,Joel M,Paulson L, Bolger AM, Lohse M&Usadel B.Trimmomatic: aflexibletrimmerfor Lekva T, Berg JP, Heck A,Lyngvi Fougner S,Olstad OK,Ringstad G, Lekva T, Berg JP, Lyle R, Heck A,Ringstad G,Olstad OK, Fougner SL, Lekva T, Borota OC,Hald JK,Bollerslev J&Berg JP. Evang JA, Berg JP, Casar-Borota O,Lekva T, Kringen MK,Ramm- Thiery JP, Acloque H,Huang RY &Nieto MA.Epithelial- Feng J, Yu SY, Li CZ,Li ZY&Zhang YZ. Integrativeproteomicsand Lee M, Marinoni I,Irmler M,Psaras T, Honegger JB,Beschorner R, Li J, Li C,Wang J, Song G,Zhao Z,Wang H, Wang W, Li H,Li Z, Normann KR, Oystese KA,Berg JP, Lekva T, Berg-Johnsen J, Goff LA, Trapnell C &Kelley D.CummeRbund:visualizationand Trapnell C, Roberts A,Goff L,Pertea G,Kim D,Kelley DR, Langmead B &Salzberg SL.Fastgapped-readalignmentwith Kim D, Pertea G,Trapnell C, Pimentel H,Kelley R&Salzberg SL. Thiede B, Grieg Z,Ramsnes I,Skjellegrind HK (https://doi.org/10.1016/j.mce.2016.08.030) Illumina sequencedata. pone.0066927) recovery. treatment withsomatostatinanalogsisassociatedpoorclinical presence ofEMTprogressioninsomatotrophadenomasfollowing Bollerslev J &Ueland T. AttenuatedRORCexpressioninthe org/10.1210/en.2013-1051) adenomas. splicing regulatorprotein1andalternativeinsomatotroph Michelsen AE, Casar-Borota O,Bollerslev J&Ueland T. Epithelial 2334–2342. response. is relatedtotumorsize,invasiveness,andsomatostatinanalog adenomas The expressionofE-cadherininsomatotrophpituitary 2265.2011.04109.x) Endocrinology tumours. with progressionofcorticotrophpituitary Pettersen J &Bollerslev J.ReducedlevelsofE-cadherincorrelate 871–890. mesenchymal transitionsindevelopmentanddisease. MMP9 signaling pathway is correlated with invasion of pituitary MMP9 signalingpathwayiscorrelated withinvasionofpituitary transcriptomics revealedthatactivation oftheIL-6R/JAK2/STAT3/ 2013 gonadotrophadenomas. of humanpituitary identifies novelmolecularmechanisms involvedinthepathogenesis Transcriptome adenomas analysisofMENX-associatedratpituitary Anastasov N, Beckers J,Theodoropoulou M,Roncaroli F oncotarget.13948) RNA-seq. gonadotrophinadenomasby lncRNAs andmRNAsinprimary Miao Y 8 multipotent stemcellsfromtheadulthumanbrain. adenomas. reference genesforRT-qPCR analysisinalargecohortofpituitary Bollerslev J &Olarescu NC.Selectionandvalidationofreliable vignettes/cummeRbund/inst/doc/cummeRbund-manual.pdf (available at: exploration ofcufflinkshigh-throughputsequencingdata.2014. org/10.1038/nprot.2012.016) TopHat andCufflinks. and transcriptexpressionanalysisofRNA-seqexperimentswith Pimentel H, Salzberg SL,Rinn JL&Pachter L.Differentialgene nmeth.1923) Bowtie 2. (https://doi.org/10.1186/gb-2013-14-4-r36) insertions, deletionsandgenefusions. TopHat2: accuratealignmentoftranscriptomesinthepresence doi.org/10.1093/bioinformatics/btu170) e71334. 126 et al PLoS ONE 137–150. (https://doi.org/10.1016/j.cell.2009.11.007) Oncotarget Nature Methods Journal ofClinicalEndocrinologyand Metabolism Journal (https://doi.org/10.1371/journal.pone.0071334) Endocrinology Molecular andCellularEndocrinology . Genome-wideanalysisofdifferentiallyexpressed (https://doi.org/10.1210/jc.2009-2197) 2011 http://bioconductor.org/packages/release/bioc/ 75 2013 2017 (https://doi.org/10.1007/s00401-013-1132-7) 811–818. Nature Protocols 2013 Bioinformatics 2012 8 8 e66927. Downloaded fromBioscientifica.com at10/02/202112:15:28PM 4585–4606. 154 9 (https://doi.org/10.1111/j.1365- 357–359. 3331–3343. (https://doi.org/10.1371/journal. 2012 2014 Genome Biology (https://doi.org/10.18632/ (https://doi.org/10.1038/ 178 7 30 Acta Neuropathologica 562–578. et al 2016 2114–2120. :3 (https://doi. . Expansionof Clinical 437 PLoS ONE 2013 Cell (https://doi. et al 2010 183–189. 2009 (https:// 14 ). . 2013 306 95 R36. 139

via freeaccess

European Journal of Endocrinology

38 37 36 35 34 33 32 31 Clinical Study Park SY, Choi M,Park D,Jeong M,Ahn KS,Lee J,Fisher PB,Yun M Wang Z, Tang ZY, Yin Z, Wei YB, Liu LF, Yan B, Zhou KQ,Nian YQ, Zahr A, Alcaide P, Yang J, delaPaz NG,Patel- Jones A,Gregory M, Gu C, Feng L,Peng H,Yang H, Feng Z&Yang Y. MTDHisan Hu G, Wei Y &Kang Y. ThemultifacetedroleofMTDH/AEG-1in Raverot G, Burman P, McCormack AI,Heaney AP, Petersenn S, Di Ieva A,Rotondo F, Syro LV, Cusimano MD&Kovacs K.Aggressive Attar M, Newey PJ, Nesbit MA,Rimmer AJ,Head RA,Gorvin CM, & Lee SG.AEG-1promotesmesenchymaltransitionthroughthe (https://doi.org/10.2147/OTT.S104556) transition incarcinoma. Gao YL &Yang JR. Metadherinregulatesepithelial-mesenchymal 10363. conditions. resting andinflammatory leukocyte-endothelial celladhesionandhasacriticalroleunder Hett S, Nevers T, Koirala A,Luscinskas FW oncotarget.6610) treatment. inmultiplemyeloma,whichissuppressedbyBortezomib (https://doi.org/10.1158/1078-0432.CCR-09-0049) cancer progression. 0796) Endocrinology tumoursandcarcinomas.aggressive pituitary Endocrinology clinicalpracticeguidelinesforthemanagementof Popovic V, Trouillas J &Dekkers O.EuropeanSocietyof nrendo.2014.64) Reviews Endocrinology adenomas–diagnosisandemergingtreatments. pituitary doi.org/10.1210/jc.2012-4028) Clinical EndocrinologyandMetabolism adenomas. sequencing studiesofnonfunctioningpituitary Wass JA,Gregory L, Buck D,Karavitaki N 195–203. null celladenomas. (https://doi.org/10.1038/ncomms10363) (https://doi.org/10.1016/j.mce.2016.07.025) Oncotarget 2018 178 Clinical CancerResearch Molecular andCellularEndocrinology 2014 2016 G1–G24. OncoTargets andTherapy 10 7 4559–4569. 423–435. (https://doi.org/10.1530/EJE-17- 2013 C MFalch Nature Communications (https://doi.org/10.1038/ (https://doi.org/10.18632/ et al et al 98 2009 E796–E800. . Whole-exome European Journal of European Journal . Endomucinprevents 2016 15 et al. 5615–5620. 9 2016 2429–2436. Nature (https:// Journal of Journal 2016 436

7

Accepted 19December2017 Revised versionreceived16December2017 Received 23August2017

gonadotroph NFPAs Fast- andslow-growing 45 44 43 42 41 40 39 Raverot G, Jouanneau E&Trouillas J. Managementofendocrine Kim SY, Yasuda S, Tanaka H, Yamagata K &Kim H.Non-clustered Alvarez Julia A,Frasch AC&Fuchsova B.Neuronalfilopodium Zhou W &Thiery JP. LossofGit2inducesepithelial-mesenchymal Li S, Wang L, Zhao Q,Liu Y, He L,Xu Q,Sun X,Teng L, Cheng H Ikeuchi Y, Dadakhujaev S,Chandhoke AS,Huynh MA,Oldenborg A, Knosp E, Steiner E, Kitz K & Matula C. Pituitary adenomaswith Knosp E, Steiner E,Kitz K&Matula C.Pituitary doi.org/10.4161/cam.5.2.14374) protocadherin. jnc.13552) ofNeurochemistry Journal facilitated bycoronin-1a,Rac1,andp21-activatedkinase1(Pak1). formation inducedbythemembraneglycoproteinM6a(Gpm6a)is jcs.126367) ofCellScience Journal transition bymiR146a-Cnot6L-controlledexpressionofZeb1. (https://doi.org/10.1074/jbc.M113.546077) Hook1. mesenchymal transitionmodulatedbyitsnovelinteractingprotein & Ke Y. SHP2positivelyregulatesTGFbeta1-inducedepithelial- 25067–25078. regulator SnoN1. small ubiquitin-likemodifier(SUMO)E3ligaseforthetranscriptional protein regulatesepithelial-mesenchymaltransitionbyoperatingasa Ikeuchi M, Deng L,Bennett EJ,Harper JW, Bonni A Reports activation ofRhoGTPasesinhumanglioblastomacells. 00008) 1993 imaging classificationcomparedwithsurgicalfindings. invasion ofthecavernoussinusspace:amagneticresonance org/10.1530/EJE-13-1031) ofEndocrinology European Journal tumoursforpersonalizedtherapeuticstrategies. of pituitary disease: clinicopathologicalclassificationandmolecularmarkers 33 2016 Journal ofBiologicalChemistry Journal 610–617. 36 (https://doi.org/10.1074/jbc.M114.575878) Cell AdhesionandMigration 2641–2646. Journal ofBiologicalChemistry Journal (https://doi.org/10.1227/00006123-199310000- 2013 2016 126 Downloaded fromBioscientifica.com at10/02/202112:15:28PM (https://doi.org/10.3892/or.2016.5106) 137 2740–2746. 2014 46–61. 2014 170 2011 R121–R132. (https://doi.org/10.1111/ 289 178 (https://doi.org/10.1242/ 2014 www.eje-online.org 34152–34160. :3 5 97–105. et al 289 (https://doi. . TIF1gamma Oncology

Neurosurgery

(https:// 307 via freeaccess