BAZZOCCO_PhD_THESIS.pdf

Identification of novel therapeutic targets
and tumor suppressor genes in colon cancer using
genome-wide high-throughput approaches
Sarah Bazzocco
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TableofContents
I
TableofContent
TABLEOFCONTENT.........................................................................................................................I
INDEXOFFIGURES.........................................................................................................................IV
INDEXOFTABLES............................................................................................................................V
ABSTRACT.....................................................................................................................................VI
1INTRODUCTION.......................................................................................................................1
1.1THEDIGESTIVETRACT...................................................................................................................1
1.2THEHUMANINTESTINE.................................................................................................................2
1.2.1Anatomyofthesmallintestine.......................................................................................2
1.2.2Histologyoftheepitheliumofthesmallintestine...........................................................4
1.2.3Anatomyandhistologyofthelargeintestine.................................................................6
1.2.4Homeostasisofthehumanintestinalepithelium............................................................6
1.2.4.1Proliferationanddifferentiationofintestinalstemcells............................................7
1.2.4.2Signalingintheintestinalstemcells...........................................................................8
1.3GENERALCHARACTERISTICSOFCANCER.........................................................................................10
1.3.1Typesofcancer..............................................................................................................10
1.3.2WorldandEuropeancancerstatistic............................................................................11
1.3.3Hallmarksofcancer.......................................................................................................13
1.3.4Tumorsuppressorgenesandoncogenes......................................................................16
1.3.5Epigeneticalterationsincancer....................................................................................18
1.4COLORECTALCANCER.................................................................................................................19
1.4.1Originandclinicalclassificationofcolorectalcancer....................................................19
1.4.2Incidenceandstatisticsofcolorectalcancer.................................................................22
1.4.3Molecularpathwaysincolorectalcancer......................................................................24
1.4.3.1Themultistepnaturefromnormalepitheliumtocolorectalcancer........................24
1.4.3.2Thedistinctpathstocolorectalcancer.....................................................................25
1.4.3.3Microsatelliteinstability(MSI)‐DNAmismatchrepairdefectsincolorectalcancer26
1.4.3.4–Chromosomalinstability(CIN)/microsatellitestability(MSS)‐GeneticInstabilityin
colorectalcancer.......................................................................................................................28
1.4.3.5CpGislandmethylatorphenotype(CIMP)‐Epigeneticchangesincolorectalcancer
28
1.4.3.6Somaticoncogeneandtumorsuppressorgenemutationsincolorectalcancer......29
1.4.4Treatmentofcolorectalcancer.....................................................................................33
1.4.5Treatmentofcoloncancerbystage..............................................................................33
1.4.5.1Chemotherapeuticagentsavailableforcolorectalcancertreatment......................34
1.4.5.2Strategiestoimprovetheobjectiveresponserateinchemotherapyforcolorectal
cancerpatients..........................................................................................................................36
2AIMSOFTHESTUDY..............................................................................................................40

Results
II
3MATERIALSANDMETHODS..................................................................................................41
3.1MATERIALSUSEDINTHISSTUDY...................................................................................................41
3.1.1Humancolorectalcancercelllines................................................................................41
3.1.2Primarycolorectaltumorsamples.................................................................................43
3.1.3Antibodies......................................................................................................................43
3.1.4Primers...........................................................................................................................44
3.2METHODSUSEDINTHISSTUDY.....................................................................................................45
3.2.1Microarraydata.............................................................................................................45
3.2.1.1mRNAexpressionmicroarrayanalysis......................................................................45
3.2.1.2DNAmethylationmicroarrayanalysis.......................................................................45
3.2.2Identificationofgenes...................................................................................................46
3.2.2.1AssociationsbetweenmRNAexpressionandthedoublingtimeofcelllines...........46
3.2.2.2AssociationsbetweenmRNAexpressionandpromotermethylationlevels............46
3.2.3Functionalgroupenrichmentanalysis...........................................................................46
3.2.4Invitroexperiments.......................................................................................................47
3.2.4.1Determinationofthedoublingtime.........................................................................47
3.2.4.2Proliferationassay.....................................................................................................47
3.2.4.3Growthinhibitionassay.............................................................................................48
3.2.4.4Apoptosisandcellcycleanalysis...............................................................................48
3.2.4.5Clonogenicassay........................................................................................................49
3.2.4.6RNAiknockdownofPPOXandGAPDH......................................................................49
3.2.4.7ProteinextractionandWesternblot.........................................................................49
3.2.4.8RNAextractionandquantitativeRT‐PCR..................................................................49
3.2.4.9DNAextractionandbisulfitesequencing..................................................................50
3.2.5ZNF238overexpressionincoloncancercells.................................................................50
3.2.5.1ZNF238cloning..........................................................................................................50
3.2.5.2ZNF238overexpressionincoloncancercells............................................................50
3.2.6Invivoanimalexperiments............................................................................................51
3.2.6.1Drugeffectsinvivousingaxenograftmodel............................................................51
3.2.6.2Determinationofthegradeofdifferentiationofcelllinesinaxenograftmodel.....51
3.2.6.3TheeffectofoverexpressingZNF238incellsinvivousingaxenograftsmodel.......51
4RESULTS................................................................................................................................52
4.1HIGHLYEXPRESSEDGENESINRAPIDLYPROLIFERATINGTUMORCELLSASNEWTARGETSFORCOLORECTAL
CANCERTREATMENT...............................................................................................................................52
4.1.1Proliferationofcolorectalcancercelllines....................................................................52
4.1.2Expressionprofilingofcolorectalcancercelllineswithdifferentgrowthrates............55
4.1.3IdentificationofPPOXandGAPDHasnewcandidatetherapeutictargets...................63
4.1.4InhibitionofPPOXandGAPDHreducesthegrowthofcolorectalcancercellsinvitro.66
4.1.5PPOXinhibitionreducesthegrowthofcoloncancercellsinaxenograftmodel..........73
4.2EXPRESSIONREGULATIONBYEPIGENETICSILENCING.........................................................................75
4.2.1Genome‐wideanalysisofCpGmethylationincolorectalcancercelllines....................75