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Identification and characterization of genes and signaling pathways involved in proliferation and differentiation of mammary epithelial cells [Elektronische Ressource] / by Marcin Jankiewicz

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IdentificationandcharacterizationofgenesandsignalingpathwaysinvolvedinproliferationanddifferentiationofmammaryepithelialcellsDissertationThesissubmittedinfulfillmentoftherequirementsforthedegreeDoctorofPhilosophyatJohannWolfgangGoetheUniversityinFrankfurtamMainByMarcinJankiewiczborninPoznan,PolandFrankfurtamMain2006ACKNOWLEDGMENTSI would like to thank Professor Dr. Bernd Groner for giving me the opportunity tocometoGermanyandtoworkonaveryimportantandinterestingprojectinhisgroup.I greatly acknowledgeourfruitfulldiscussionsandhissupportduringcompletionofmythesis.IwouldliketothankProfessorDr.AnnaStarzinski-Powitzforherinterestinmyworkandhersupervisionduringcompletionofmythesis.Many thanks to all members of the Groner group: Corinna Bähr, Nadja Bednorz,Andrea Belaus, Nadine Böcher, Boris Brill, Claudia Bürger, Natalia Delis, SylaneDesrivieres, Maresa Eck, Christina Gewinner, Corina Heinz, Sabrina Kraemer,ChristianKunz,NahomiCastro-Palomino-Laria,CarrieShemanko,VidaVafaizadeh,AstridWeiss,IlkaWittigandKerstinNagel-Wolfrum.Ienjoyedtheextraordinarytimewespendtogetherinthelab,inourmeetingsandatcrazyparties.

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Published 01 January 2008
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Identificationandcharacterizationof
genesandsignalingpathwaysinvolvedin
proliferationanddifferentiationof
mammaryepithelialcells


Dissertation

Thesissubmittedinfulfillmentofthe
requirementsforthedegree

DoctorofPhilosophy
at
JohannWolfgangGoetheUniversity
inFrankfurtamMain

By
MarcinJankiewicz
borninPoznan,Poland



FrankfurtamMain
2006

ACKNOWLEDGMENTS

I would like to thank Professor Dr. Bernd Groner for giving me the opportunity to
cometoGermanyandtoworkonaveryimportantandinterestingprojectinhisgroup.
I greatly acknowledgeourfruitfulldiscussionsandhissupportduringcompletionof
mythesis.

IwouldliketothankProfessorDr.AnnaStarzinski-Powitzforherinterestinmywork
andhersupervisionduringcompletionofmythesis.

Many thanks to all members of the Groner group: Corinna Bähr, Nadja Bednorz,
Andrea Belaus, Nadine Böcher, Boris Brill, Claudia Bürger, Natalia Delis, Sylane
Desrivieres, Maresa Eck, Christina Gewinner, Corina Heinz, Sabrina Kraemer,
ChristianKunz,NahomiCastro-Palomino-Laria,CarrieShemanko,VidaVafaizadeh,
AstridWeiss,IlkaWittigandKerstinNagel-Wolfrum.Ienjoyedtheextraordinarytime
wespendtogetherinthelab,inourmeetingsandatcrazyparties.

IwouldliketomentionDr.SylaneDesrivieresandthankherforourlongdiscussions
onmyresultsandhelpfulcriticismofmythesismanuscript.

I also would like to thank Dr. Corina Heinz for her good advice and especially for
criticalreadingofmythesismanuscript.

Idon’twanttoforgetmyfriendsworkinginothergroupsattheGSHandthankthem
forthejoyfultimeandnicecollaborationsthatIhadwiththem.

Many thanks to my parents and Alicja. They always supported me in good and bad
timesduringmystudy.

Intheend,IwouldlikethanktoProf.Dr.hab.AnnaC.Majewska,ProfDr.hab.Jan
Sadowski,Prof.Dr.hab.ArturJarmołowski,Dr.WojciechG.Musiałfortheirhelpand
supporttofindagraduatestudentposition.

2
ABSTRACT
The mammary gland is a perfect system to study the pathways regulating
organogenesis during development of an individual. The proper development of the
mammaryglandrequiresatightcoordinationofexpressionofmanygenesinvolvedin
proliferationanddifferentiation.Theaimofthisworkwastoidentifynovelgenesand
pathways involved in the development of the mammary gland and to find possible
correlations between the signaling pathways and their downstream targets that are
activated during proliferation and functional differentiation of mammary epithelial
cells.InthisstudyrapamycinhasbeenusedtoinhibitthemTORproteintoanalyzeits
role during mammary gland development. Further a genomic approach was used to
identifygenesdifferentlyexpressedduringthisprocess.
TheanalysisoftheeffectscausedbytheinhibitionofthemTORsignalingpathwayby
usingrapamycinonmammaryepithelialcellsforthefirsttimedemonstratethatmTOR
plays central role in the coordination of pathways governing the proliferation and
differentiationofepithelialcellsduringmammaryglanddevelopment.Moredetailed
analysisledtotheidentificationofId1andId2astwomajordownstreameffectorsof
themTORsignalingpathwayregulatingproliferationanddifferentiation,respectively.
Thegenomicsanalysisrevealedseveralinterestinggenesinvolvedintheregulationof
aproliferativeorsecretoryphenotypeofnormalepithelialcellsin vitro.Variousgenes
identified by microarray analysis are of high interest and to determine their role in
mammaryglanddevelopment.Amongtheidentifiedgenessomecontributetoprocess
of proliferation like Nol5 and Kpna2, whereas other genes are required for proper
functional differentiation such as Nkd2 and Cited4. Importantly, the mentioned
candidategenesarealsointerestingregardingcancerdevelopment,sincederegulation
oftheirexpressionmigthcontributetotumorformation.
Thefindingsdescribedinthisworkclearlycontributetoourbetterunderstandingof
the mTOR signaling pathway regulating expression of the genes involved in the
development of mammary gland. In additon, the presented results should allow
broadening our view of the events that contribute to breast cancer development and
helptodesignbetteranticancertherapiesinthefuture.


3Contents
CONTENTS
ACKNOWLEDGMENTS........................................................................2
ABSTRACT...............................................................................................3
CONTENTS ..............................................................................................4
1.INTRODUCTION ..............................................................................10
1.1Developmentofthemousemammarygland .................................................10
1.1.1Embryonaldevelopmentofthemammarygland .........................................10
1.1.2Developmentofthemammaryglandduringpuberty ..................................11
1.1.3Developmentofthesecretorycompartmentduringpregnancy ...................12
1.1.4Lactationinthemammarygland..................................................................13
1.1.5Involutionofthemammarygland................................................................13
1.2 Signaling pathways playing a essential roles during mammary gland
development ............................................................................................................15
1.2.1Prolactin/Stat5signalinginthemammarygland .........................................15
1.2.2Theinsulin/IGFpathwayinmammaryglanddevelopment.........................17
1.3TargetofRapamycin .......................................................................................20
1.3.1DiscoveryofTOR ........................................................................................20
1.3.2StructureofmTOR.......................................................................................20
1.3.3ThefunctionofthemTORprotein...............................................................22
1.3.4RegulationofmTORactivity.......................................................................23
1.3.4.1ThePI3K/AktsignalingpathwayisanupstreamregulatorofmTOR..23
1.3.4.2TSCnegativelyregulatesmTOR ..........................................................24
1.3.4.3RhebapositiveregulatorofmTORissuppressedbyTSC...................24
1.3.4.4AMPKactivatesTSC2andnegativelyregulatesthemTORpathway..26
1.3.4.5RegulationofTSCby14-3-3proteins ..................................................26
1.3.5ComponentsofthemTORcomplex.............................................................27
1.3.5.1mTORiscomplexedwithRaptorandGβL ..........................................27
1.3.5.2RictorisacomponentofthemTORcomplex.......................................29
1.3.6DownstreameffectorsofthemTORpathway..............................................31
1.3.6.1Phosphorylationof4E-BP1bymTORleadstotheactivationofeIF4E
...........................................................................................................................32
1.3.6.2mTORphosphorylatesS6kinasepromotingtranslation......................33
1.3.6.3mTORregulatestheeIF4BthroughS6K..............................................34
1.3.6.4mTORactivatesdegradationofPDCD4throughS6kinase.................34
4Contents
1.3.6.5PhosphorylationloopsinthemTOR/S6Ksignalingpathway ..............35
1.4ThemTORpathwayandcancerdevelopment..............................................36
1.5TheroleofbHLHproteinsinmammaryglanddevelopment......................37
1.5.1TheroleofId1inproliferationandinvasion ...............................................39
1.5.2TheroleofId2duringpregnancyandlactation ...........................................40
1.6RNAinterferenceasatoolfortheanalysisofgenesinvolvedinmammary
glanddevelopment..................................................................................................41
1.6.1EndogenousexpressionofsiRNA................................................................42
1.6.2 The use of lentiviral vectors for stable siRNA expression in mammalian
cells........................................................................................................................43
1.7Systemsforcultivatingmammaryepithelialcells.........................................44
1.8Theaimsofthiswork.......................................................................................46
2MATERIALSANDMETHODS............................. ...........................47
2.1MATERIALS....................................................................................................47
2.1.1Chemicals.....................................................................................................47
2.1.2Commercialkitsandreagents ......................................................................48
2.1.3Enzymes .......................................................................................................48
2.1.4Materialusedincellculture.........................................................................48
2.1.5Markers.........................................................................................................49
2.1.6Plasmids .......................................................................................................49
2.1.7Antibodies ....................................................................................................50
2.1.8Primers .........................................................................................................50
2.1.9Labware........................................................................................................52
2.1.10Eukaryoticcelllines...................................................................................53
2.1.11Bacterialstrains..........................................................................................53
2.1.12Culturemedia .............................................................................................53
2.1.13Buffersandsolutions..................................................................................55
2.2METHODS .......................................................................................................56
2.2.1MOLECULARBIOLOGYMETHODS......................................................56
2.2.1.1Preparationoftransformationcompetentbacterialcells.......................56
2.2.1.2Transformationofbacterialcells...........................................................56
2.2.1.3PlasmidDNAMiniPreparation ............................................................57
2.2.1.4PlasmidDNAMaxiPreparation ...........................................................57
2.2.1.5DNAconcentrationmeasurement .........................................................58
5Contents
2.2.1.6RNAconcentrationmeasurement .........................................................58
2.2.1.7Sequencingofnucleicacids ..................................................................59
2.2.1.8GelElectrophoresisofDNA .................................................................59
2.2.1.9ExtractionofDNAfragmentsfromagarosegels..................................60
2.2.1.10DigestionofDNAwithrestrictionendonucleaseenzymes ................60
2.2.1.11Dephosphorylationof5'endsofDNA................................................61
2.2.1.12LigationofDNAfragmentsintoplasmids..........................................61
2.2.1.13FasttotalRNApurificationfromculturedcells..................................62
2.2.1.14ReverseTranscriptionofmRNA ........................................................63
2.2.1.15Real-TimePCR ...................................................................................63
2..2.1.16AdditionofrestrictionsitestoclonedsequenceusingPCR ..............66
2.2.1.17ExtractionoftotalRNAfrommammaliancell...................................66
2.2.18IsolationofRNAandproteinsfromtissues...........................................67
2.2.2VIROLOGYMETHODS.............................................................................68
2.2.2.1Generationofviralparticles..................................................................68
2.2.2.2Titrationoflentiviralparticlescontainingsupernatants........................69
2.2.2.3Transductionofepithelialcellswithlentiviralparticles .......................70
2.2.3PROTEINBIOCHEMISTRYMETHODS..................................................70
2.2.3.1IsolationofproteinsusingRIPA-Buffer ...............................................70
2.2.3.2Determinationoftheproteinconcentration ..........................................71
2.2.3.3SDS-polyacrylamid-gel-electrophorese ................................................71
2.2.3.4Electro-transferofproteinsontonitrocellulosemembranes .................72
2.2.3.5Verificationofproteintransferonnitrocellulosemembranes ..............72
2.2.3.6Detectionofproteinsonawesternblot.................................................72
2.2.3.7Strippingofnitrocellulosemembranes .................................................73
2.2.3.8Preparationofnuclearproteinextracts..................................................73
2.2.4CELLCULTUREMETHODS ....................................................................74
2.2.4.1Invitrodifferentiationofmammaryepithelialcells .............................74
2.2.4.2Splittingmammalianadherentcells ......................................................74
2.2.4.3Isolationandcultureofprimarymammaryepithelialcellsfrommice.75
2.2.4.4Freezingandthawingmammaliancells................................................76
2.2.4.5Three-dimensionalcultureofmammaryepithelialcells.......................77
2.2.4.6Proliferationassay.................................................................................78
2.2.4.7Luciferase-Assay...................................................................................78
6Contents
2.2.4.8TransfectionofmammaliancellswithLipofectamine..........................79
2.2.4.9TransfectionwithPolyethylenimine .....................................................79
2.2.4.10TransfectionwithCalciumPhosphate.................................................80
2.2.5HISTOCHEMICALMETHODS.................................................................81
2.2.5.1Preparationofmammaryglandwholemounts .....................................81
2.3INSTRUMENTATION....................................................................................82
3.RESULTS ............................................................................................83
3.1TheroleofmTORsignalinginmammaryepithelialcells............................83
3.1.1ActivationofmTORinproliferatinganddifferentiatingHC11cells..........84
3.1.2 Influence of mTOR on expression ofβ-casein during differentiation of
mammaryepithelialcells ......................................................................................85
3.1.3Effectofrapamycinonβ-caseinmRNAstability........................................87
3.1.4InfluenceofmTORonthemorphologyofmammaryepithelialcells .........88
3.1.4.1ThemorphologyofHC11cellsin2Dand3Dcultures.........................88
3.1.4.2InfluenceofmTORonthemorphologyofHC11cellsin3Dculture...89
3.1.5InfluenceofmTORinhibitionontheproliferationofHC11cells...............90
3.1.6TheinfluenceofmTORontheStat5signalingpathway.............................91
3.1.6.1TheinfluenceofmTORonthephosphorylationofStat5.....................92
3.1.6.2InfluenceonnucleartranslocationofStat5...........................................93
3.1.6.3InfluenceontranscriptionalactivationofStat5 ....................................94
3.1.7TheInfluenceofmTORontheexpressionofIdbproteins..........................97
3.1.7.1RegulationofId1bymTOR .................................................................97
3.1.7.2.RegulationofId2expressionbymTOR. .............................................98
3.1.8Theinfluenceofrapamycinonprimarymammaryepithelialcells ...........100
3.2TheroleofmTORduringdevelopmentofthemammarygland...............102
3.2.1RoleofmTORduringpregnancy...............................................................102
3.2.2TheroleofmTORduringlactation...........................................................103
3.2.2.1Theinfluenceofrapamycinonthedevelopmentofpups ...................104
3.2.2.2EffectofmTORinhibitiononglandweightandmilkproduction......105
3.2.2.3Morphologyofthemammaryglandofrapamycintreatedmice.........106
3.2.2.4TheeffectofrapamycinonexpressionofId2andβ-casein. ..............107
3.3 The effect of rapamycin on mammary epithelial cells over4expressing Id
proteins..................................................................................................................109
3.3.1Preparationofplasmidsfortheproductionoflentiviruses ........................110
7Contents
3.3.2Transfectionof293Tcellsforthegenerationofthelentiviralparticles....111
3.3.3ViraltransductionofHC11cellsandover-expressionofIdbproteins......112
3.3.4 The effect of Id1 over-expression on proliferation and differentiation of
HC11cells...........................................................................................................113
3.3.5BranchingofHC11cellsover-expressingId1 ...........................................116
3.3.6 Effect of Rapamycin on the functional differentiation of HC11 over-
expressingId2 .....................................................................................................118
3.4Identificationofgenesinvolvedinproliferationanddifferentiationofmouse
mammaryepithelialcells.....................................................................................119
3.4.1Micro-arrayanalysisofgeneexpressionduringdifferentiationofmammary
epithelialcells......................................................................................................119
3.4.2Confirmationofmicro-arraydatabyReal-Time-PCRanalysis. ...............120
3.4.3SilencingofcandidategenesbyRNAinterference ...................................122
3.4.3.1 Preparation of lentiviral vectors with siRNA targeting Id1 and Id2
mRNA .............................................................................................................123
3.4.3.2Transfectionofpackagingcellswithlentiviralvectors ......................125
3.4.3.3StabletransductionofHC11cellswithlentiviralvectors...................126
3.4.4DifferentiationofHC11cellsexpressingsiRNAagainstId2....................128
3.4.5MorphologicaldifferentiationofHC11cellsexpressingsiRNAtargetingId1
.............................................................................................................................129
3.4.6ProliferationofHC11expressingsiRNAsilencingId1.............................130
4.DISCUSSION....................................................................................132
4.1ThemTORpathwayplaysaroleinproliferationanddifferentiationofthe
mammarygland....................................................................................................132
4.1.1HC11cellsasamodelsystemtostudythemTORpathway .....................132
4.1.2Morphologicaldifferentiationofmammaryepithelialcellscanbestudiedin
3Dculture............................................................................................................133
4.1.3ThemTORpathwaycontrolstheproliferationofmammaryepithelialcells
.............................................................................................................................134
4.1.4 The mTOR pathway is essential for the functional differentiation of
mammaryepithelialcells ....................................................................................135
4.1.4.1Rapamycininhibitsβ-caseinexpressionin vitro ................................135
4.1.4.2IntactmTORsignalingisessentialduringmammaryglanddevelopment
.........................................................................................................................136
8Contents
4.1.4.3ThemTOR-dependentexpressionofβ-caseinisnotmediatedbyStat5
signaling. .........................................................................................................137
4.1.5 mTOR controls morphological and functional differentiation of mammary
epithelialcellsthroughIdbproteins....................................................................138
4.1.5.1mTORregulatesIdbexpressiononthemRNAlevel..........................139
4.1.5.2PotentialtranscriptionalregulationofIdbproteinsbyC/EBPβ .........139
4.1.5.3PotentialregulationofIdbproteinsbyHIF ........................................141
4.1.6RoleofmTORandIdbproteinsincancer .................................................143
4.1.7ConclusionsandOutlook ...........................................................................144
4.2Identificationofgenesinvolvedinmammaryglanddevelopmentmayhelp
toidentifyfactorsessentialforbreastcancer ....................................................145
4.2.1 The use of microarrays allowed the identification of differently expressed
genes....................................................................................................................146
4.2.2Selectionofcandidategenes ......................................................................147
4.2.3 Characterization and functional analysis of genes identified in the
microarray ...........................................................................................................147
4.2.3.1Nkd2....................................................................................................148
4.2.3.2Nol5.....................................................................................................150
4.2.3.3Kpna2 ..................................................................................................151
4.2.3.4Cited4 ..................................................................................................152
4.2.4ConclusionsandOutlook ...........................................................................154
REFERENCES .....................................................................................155
ABBREVIATIONS...............................................................................172
ZUSAMMENFASSUNG......................................................................175
PUBLICATIONS..................................................................................183
CURRICULUMVITAE ......................................................................184
DECLARATION ..................................................................................186








9Introduction
1.INTRODUCTION
The development of all life forms starts from a single cell. The formation of
complicated tissue structures and organs in higher organized organism requires
multiple cell divisions. This process also depends on coordinated signaling between
single cells as well as between the thousands of cells forming a tissue structure or
organ.Theproliferationandfunctionaldifferentiationofallcellsneedstobetightly
controlled, since deregulation of this process may lead to tumor formation.
Investigatingthegenes,proteinsandsignalingpathwaysthatcontrolcellproliferation
and differentiation will increase our understanding of how its deregulation leads to
disease and cancer development. To study these genes, proteins and signaling
pathways,whichcontrolorganogenesis,themousemammaryglandisaveryattractive
model.Thedevelopmentofthemammaryglandoccursmainlyafterbirthandunlike
most other organs, the majority of the studies on developmental regulation can
thereforebeperformedinjuvenileoradultorganisms(Medina,1996).
1.1Developmentofthemousemammarygland
Thedevelopmentofthemousemammaryglandincludingthecyclesofproliferation,
functionaldifferentiationandapoptosisofthesecretoryepitheliumwhichoccurwith
eachpregnancyistightlycontrolledbyhormones(HennighausenandRobinson,2005).
To understand the various signaling events occurring during proliferation and
differentiation of the epithelial cells the anatomy and development of the mammary
glandwillbedescribedinmoredetail.
1.1.1Embryonaldevelopmentofthemammarygland
In mice, embryonal development of the mammary glands starts at day 10-11 after
fertilization of the egg. Five pairs of embryonic mammary buds are formed after
invagination of embryonic epithelial cells at ten positions along the so called “milk
line”. The milk line is a single layer of ectoderm with enlarged cells positioned
bilaterally and extending from the anterior to the posterior limb. At day E12 the
mammaryepitheliumconsistsofseveralcelllayersandhasashapeofalightbulb.At
thispointitissurroundedbyaspecializedfibroblastlayerknownasdensemammary
mesenchyme derived from the subdermis. In male embryos, the fetal testes start to
produceandrogensondayE13causingthedestructionofthemammarybuds.
10