<p>HER2 Gene-Protein Assay (GPA) је посебно погодан за истовремено процењивање експресије HER2 протеина и статуса амплификације HER2 гена на нивоу појединачних ћелија и њихово повезивање са ћелијском морфологијом. Циљ истраживања био је испитати да ли су постојећи критеријуми препоручени од стране ASCO/CAP довољни за дијагностиковање HER2 позитивности код пацијенткиња које показују интратуморску хетерогеност, како у примарним туморима тако и у метастазама у регионалне лимфне чворове, учесталост HER2 хетерогености у макрометастазама лоцираним у лимфним чворовима, те да ли постоји јасна корелација између хетерогености нађене у примарном тумору дојке и припадајућим метастазама у лимфним чворовима. Испитивање је обухватило 41 од планиране 51 пацијенткиње које су испуниле све критеријуме укључивања. Репрезентативни парафински блокови метастатских лимфних чворова одабрани су из архивираног материјала, обојени GPA методом и процењени у складу са критеријумима ASCO/CAP 2013. Анализирано је 120 ћелија у хистолошком резу сваког метастатског лимфног чвора. Статус HER2 се разликовао између примарног тумора и његових метастаза у 13,2% (5/38) случајева. Један случај HER2 позитивног примарног тумора имао је HER2 негативне метастазе, два додатна случаја са HER2 позитивним примарним тумором су имала метастазе са статусом граничне амплификације без прекомерне експресије HER2 протеина и два случаја са HER2 негативним примарним тумором су имала метастазе са статусом граничне амплификације без прекомерне експресије HER2 протеина. У 17.4% (4/23) случајева са HER2 не-амплификованим примарним тумором метастазе су постале граничне у статусу генске амплификације. Једна од четири метастазе HER2 негативног примарног тумора показала је мали фокус HER2 позитивних туморских ћелија (&lt;3% тумора). Микрохетерогеност је анализирана у 108 лимфних чворова код 38 пацијенткиња и уочена у 22 лимфна чвора, тј. код четири пацијенткиње у свим анализираним лимфним чворовима, док је код једне пацијенткиње од 4 анализирана лимфна чвора микрохетерогеност потврђена у једном лимфном чвору. На основу добијених резултата може се закључити да постојећи критеријуми препоручени од стране ASCO/CAP применом прихваћених метода нису довољни за дијагностиковање HER2 позитивности код пацијенткиња које показују интратуморску и интертуморску хетерогеност како у примарним туморима тако и у метастазама, те да постоји статистички високо сигнификантан број макрометастаза лоцираних у лимфним чворовима које показују HER2 хетерогеност и позитивна корелација између хетерогености нађене у примарним туморима и припадајућим метастазама у лимфним чворовима.</p> / <p>HER2 Gene-Protein Assay (GPA) je posebno pogodan za istovremeno procenjivanje ekspresije HER2 proteina i statusa amplifikacije HER2 gena na nivou pojedinačnih ćelija i njihovo povezivanje sa ćelijskom morfologijom. Cilj istraživanja bio je ispitati da li su postojeći kriterijumi preporučeni od strane ASCO/CAP dovoljni za dijagnostikovanje HER2 pozitivnosti kod pacijentkinja koje pokazuju intratumorsku heterogenost, kako u primarnim tumorima tako i u metastazama u regionalne limfne čvorove, učestalost HER2 heterogenosti u makrometastazama lociranim u limfnim čvorovima, te da li postoji jasna korelacija između heterogenosti nađene u primarnom tumoru dojke i pripadajućim metastazama u limfnim čvorovima. Ispitivanje je obuhvatilo 41 od planirane 51 pacijentkinje koje su ispunile sve kriterijume uključivanja. Reprezentativni parafinski blokovi metastatskih limfnih čvorova odabrani su iz arhiviranog materijala, obojeni GPA metodom i procenjeni u skladu sa kriterijumima ASCO/CAP 2013. Analizirano je 120 ćelija u histološkom rezu svakog metastatskog limfnog čvora. Status HER2 se razlikovao između primarnog tumora i njegovih metastaza u 13,2% (5/38) slučajeva. Jedan slučaj HER2 pozitivnog primarnog tumora imao je HER2 negativne metastaze, dva dodatna slučaja sa HER2 pozitivnim primarnim tumorom su imala metastaze sa statusom granične amplifikacije bez prekomerne ekspresije HER2 proteina i dva slučaja sa HER2 negativnim primarnim tumorom su imala metastaze sa statusom granične amplifikacije bez prekomerne ekspresije HER2 proteina. U 17.4% (4/23) slučajeva sa HER2 ne-amplifikovanim primarnim tumorom metastaze su postale granične u statusu genske amplifikacije. Jedna od četiri metastaze HER2 negativnog primarnog tumora pokazala je mali fokus HER2 pozitivnih tumorskih ćelija (&lt;3% tumora). Mikroheterogenost je analizirana u 108 limfnih čvorova kod 38 pacijentkinja i uočena u 22 limfna čvora, tj. kod četiri pacijentkinje u svim analiziranim limfnim čvorovima, dok je kod jedne pacijentkinje od 4 analizirana limfna čvora mikroheterogenost potvrđena u jednom limfnom čvoru. Na osnovu dobijenih rezultata može se zaključiti da postojeći kriterijumi preporučeni od strane ASCO/CAP primenom prihvaćenih metoda nisu dovoljni za dijagnostikovanje HER2 pozitivnosti kod pacijentkinja koje pokazuju intratumorsku i intertumorsku heterogenost kako u primarnim tumorima tako i u metastazama, te da postoji statistički visoko signifikantan broj makrometastaza lociranih u limfnim čvorovima koje pokazuju HER2 heterogenost i pozitivna korelacija između heterogenosti nađene u primarnim tumorima i pripadajućim metastazama u limfnim čvorovima.</p> / <p><!--[if gte mso 9]><xml> <o:DocumentProperties> <o:Author>ilija vogel</o:Author> <o:Version>16.00</o:Version> </o:DocumentProperties> <o:OfficeDocumentSettings> <o:AllowPNG/> </o:OfficeDocumentSettings></xml><![endif]--><!--[if gte mso 9]><xml> <w:WordDocument> <w:View>Normal</w:View> <w:Zoom>0</w:Zoom> <w:TrackMoves/> <w:TrackFormatting/> <w:PunctuationKerning/> <w:ValidateAgainstSchemas/> <w:SaveIfXMLInvalid>false</w:SaveIfXMLInvalid> <w:IgnoreMixedContent>false</w:IgnoreMixedContent> <w:AlwaysShowPlaceholderText>false</w:AlwaysShowPlaceholderText> <w:DoNotPromoteQF/> <w:LidThemeOther>EN-US</w:LidThemeOther> <w:LidThemeAsian>X-NONE</w:LidThemeAsian> <w:LidThemeComplexScript>X-NONE</w:LidThemeComplexScript> <w:Compatibility> <w:BreakWrappedTables/> <w:SnapToGridInCell/> <w:WrapTextWithPunct/> <w:UseAsianBreakRules/> <w:DontGrowAutofit/> <w:SplitPgBreakAndParaMark/> <w:EnableOpenTypeKerning/> <w:DontFlipMirrorIndents/> <w:OverrideTableStyleHps/> </w:Compatibility> <m:mathPr> <m:mathFont m:val="Cambria Math"/> <m:brkBin m:val="before"/> <m:brkBinSub m:val="&#45;-"/> <m:smallFrac m:val="off"/> <m:dispDef/> <m:lMargin m:val="0"/> <m:rMargin m:val="0"/> <m:defJc m:val="centerGroup"/> <m:wrapIndent m:val="1440"/> <m:intLim m:val="subSup"/> <m:naryLim m:val="undOvr"/> </m:mathPr></w:WordDocument></xml><![endif]--><!--[if gte mso 9]><xml> <w:LatentStyles DefLockedState="false" DefUnhideWhenUsed="false" DefSemiHidden="false" DefQFormat="false" DefPriority="99" LatentStyleCount="371"> <w:LsdException Locked="false" Priority="0" QFormat="true" Name="Normal"/> <w:LsdException Locked="false" Priority="9" QFormat="true" Name="heading 1"/> <w:LsdException Locked="false" Priority="9" SemiHidden="true" UnhideWhenUsed="true" QFormat="true" Name="heading 2"/> <w:LsdException Locked="false" Priority="9" SemiHidden="true" UnhideWhenUsed="true" QFormat="true" Name="heading 3"/> <w:LsdException Locked="false" Priority="9" SemiHidden="true" UnhideWhenUsed="true" QFormat="true" Name="heading 4"/> <w:LsdException Locked="false" Priority="9" SemiHidden="true" UnhideWhenUsed="true" QFormat="true" Name="heading 5"/> <w:LsdException Locked="false" Priority="9" SemiHidden="true" UnhideWhenUsed="true" QFormat="true" Name="heading 6"/> <w:LsdException Locked="false" Priority="9" SemiHidden="true" UnhideWhenUsed="true" QFormat="true" Name="heading 7"/> <w:LsdException Locked="false" Priority="9" SemiHidden="true" UnhideWhenUsed="true" QFormat="true" Name="heading 8"/> <w:LsdException Locked="false" Priority="9" SemiHidden="true" UnhideWhenUsed="true" QFormat="true" Name="heading 9"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="index 1"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="index 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="index 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="index 4"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="index 5"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="index 6"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="index 7"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="index 8"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="index 9"/> <w:LsdException Locked="false" Priority="39" SemiHidden="true" UnhideWhenUsed="true" Name="toc 1"/> <w:LsdException Locked="false" Priority="39" SemiHidden="true" UnhideWhenUsed="true" Name="toc 2"/> <w:LsdException Locked="false" Priority="39" SemiHidden="true" UnhideWhenUsed="true" Name="toc 3"/> <w:LsdException Locked="false" Priority="39" SemiHidden="true" UnhideWhenUsed="true" Name="toc 4"/> <w:LsdException Locked="false" Priority="39" SemiHidden="true" UnhideWhenUsed="true" Name="toc 5"/> <w:LsdException Locked="false" Priority="39" SemiHidden="true" UnhideWhenUsed="true" Name="toc 6"/> <w:LsdException Locked="false" Priority="39" SemiHidden="true" UnhideWhenUsed="true" Name="toc 7"/> <w:LsdException Locked="false" Priority="39" SemiHidden="true" UnhideWhenUsed="true" Name="toc 8"/> <w:LsdException Locked="false" Priority="39" SemiHidden="true" UnhideWhenUsed="true" Name="toc 9"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Normal Indent"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="footnote text"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="annotation text"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="header"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="footer"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="index heading"/> <w:LsdException Locked="false" Priority="35" SemiHidden="true" UnhideWhenUsed="true" QFormat="true" Name="caption"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="table of figures"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="envelope address"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="envelope return"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="footnote reference"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="annotation reference"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="line number"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="page number"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="endnote reference"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="endnote text"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="table of authorities"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="macro"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="toa heading"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="List"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="List Bullet"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="List Number"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="List 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="List 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="List 4"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="List 5"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="List Bullet 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="List Bullet 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="List Bullet 4"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="List Bullet 5"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="List Number 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="List Number 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="List Number 4"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="List Number 5"/> <w:LsdException Locked="false" Priority="10" QFormat="true" Name="Title"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Closing"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Signature"/> <w:LsdException Locked="false" Priority="1" SemiHidden="true" UnhideWhenUsed="true" Name="Default Paragraph Font"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Body Text"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Body Text Indent"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="List Continue"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="List Continue 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="List Continue 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="List Continue 4"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="List Continue 5"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Message Header"/> <w:LsdException Locked="false" Priority="11" QFormat="true" Name="Subtitle"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Salutation"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Date"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Body Text First Indent"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Body Text First Indent 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Note Heading"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Body Text 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Body Text 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Body Text Indent 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Body Text Indent 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Block Text"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Hyperlink"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="FollowedHyperlink"/> <w:LsdException Locked="false" Priority="22" QFormat="true" Name="Strong"/> <w:LsdException Locked="false" Priority="20" QFormat="true" Name="Emphasis"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Document Map"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Plain Text"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="E-mail Signature"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="HTML Top of Form"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="HTML Bottom of Form"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Normal (Web)"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="HTML Acronym"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="HTML Address"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="HTML Cite"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="HTML Code"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="HTML Definition"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="HTML Keyboard"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="HTML Preformatted"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="HTML Sample"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="HTML Typewriter"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="HTML Variable"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Normal Table"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="annotation subject"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="No List"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Outline List 1"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Outline List 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Outline List 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Simple 1"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Simple 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Simple 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Classic 1"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Classic 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Classic 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Classic 4"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Colorful 1"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Colorful 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Colorful 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Columns 1"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Columns 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Columns 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Columns 4"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Columns 5"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Grid 1"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Grid 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Grid 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Grid 4"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Grid 5"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Grid 6"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Grid 7"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Grid 8"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table List 1"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table List 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table List 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table List 4"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table List 5"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table List 6"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table List 7"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table List 8"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table 3D effects 1"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table 3D effects 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table 3D effects 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Contemporary"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Elegant"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Professional"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Subtle 1"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Subtle 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Web 1"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Web 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Web 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Balloon Text"/> <w:LsdException Locked="false" Priority="39" Name="Table Grid"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Theme"/> <w:LsdException Locked="false" SemiHidden="true" Name="Placeholder Text"/> <w:LsdException Locked="false" Priority="1" QFormat="true" Name="No Spacing"/> <w:LsdException Locked="false" Priority="60" Name="Light Shading"/> <w:LsdException Locked="false" Priority="61" Name="Light List"/> <w:LsdException Locked="false" Priority="62" Name="Light Grid"/> <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1"/> <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2"/> <w:LsdException Locked="false" Priority="65" Name="Medium List 1"/> <w:LsdException Locked="false" Priority="66" Name="Medium List 2"/> <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1"/> <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2"/> <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3"/> <w:LsdException Locked="false" Priority="70" Name="Dark List"/> <w:LsdException Locked="false" Priority="71" Name="Colorful Shading"/> <w:LsdException Locked="false" Priority="72" Name="Colorful List"/> <w:LsdException Locked="false" Priority="73" Name="Colorful Grid"/> <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 1"/> <w:LsdException Locked="false" Priority="61" Name="Light List Accent 1"/> <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 1"/> <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 1"/> <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 1"/> <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 1"/> <w:LsdException Locked="false" SemiHidden="true" Name="Revision"/> <w:LsdException Locked="false" Priority="34" QFormat="true" Name="List Paragraph"/> <w:LsdException Locked="false" Priority="29" QFormat="true" Name="Quote"/> <w:LsdException Locked="false" Priority="30" QFormat="true" Name="Intense Quote"/> <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 1"/> <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 1"/> <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 1"/> <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 1"/> <w:LsdException Locked="false" Priority="70" Name="Dark List Accent 1"/> <w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 1"/> <w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 1"/> <w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 1"/> <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 2"/> <w:LsdException Locked="false" Priority="61" Name="Light List Accent 2"/> <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 2"/> <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 2"/> <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 2"/> <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 2"/> <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 2"/> <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 2"/> <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 2"/> <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 2"/> <w:LsdException Locked="false" Priority="70" Name="Dark List Accent 2"/> <w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 2"/> <w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 2"/> <w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 2"/> <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 3"/> <w:LsdException Locked="false" Priority="61" Name="Light List Accent 3"/> <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 3"/> <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 3"/> <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 3"/> <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 3"/> <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 3"/> <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 3"/> <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 3"/> <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 3"/> <w:LsdException Locked="false" Priority="70" Name="Dark List Accent 3"/> <w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 3"/> <w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 3"/> <w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 3"/> <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 4"/> <w:LsdException Locked="false" Priority="61" Name="Light List Accent 4"/> <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 4"/> <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 4"/> <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 4"/> <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 4"/> <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 4"/

As proteínas p53, MDM-2, c-Kit, ErbB-2, PCNA e p-glicoproteína foram estudadas em 42 amostras de osteossarcoma ao diagnóstico, através da técnica de imunohistoquímica, e foram correlacionados estes achados com o prognóstico destes pacientes. O p-53 foi positivo em 23,1% (9/39), PCNA em 71,4% (25/35), p-glicoproteína em 40,5% (15/37), MDM-2 em 34,8% (8/23), c-kit em 67,6% (25/37) e ErbB-2 em 17,9% (7/39). Na análise univariada, a presença de metástases ao diagnóstico, a positividade de p53 e ErbB-2 influenciaram o prognóstico individualmente, entretanto, na análise multivariada, a presença de metástase ao diagnóstico revelou-se o único fator de prognóstico estatisticamente significante / We study, by imunohistochemistry technique, the proteins p53, MDM-2, c-Kit, ErbB-2, PCNA and p-glycoprotein in samples of osteosarcoma tumors at diagnosis and its correlation with the prognosis of this patients. The p-53 was positive in 23,1% (9/39), PCNA in 71,4% (25/35), p-glycoprotein in 40,5% (15/37), MDM-2 in 34,8% (8/23), c-kit in 67,8%(25/37) and ErbB-2 in 17,9% (7/39) of the samples. In the univariate analysis, the presence of metastasis at diagnosis, the positivity of p-53 and ErbB-2 influenced the prognosis individually, otherwise, in the multivariate analysis, the presence of metastasis at diagnosis was the only prgnostic factor statistically significant

La signalisation par l’estrogène a longtemps été considérée comme jouant un rôle critique dans le développement et la progression des cancers hormono-dépendants tel que le cancer du sein. Deux tiers des cancers du sein expriment le récepteur des estrogènes (ER) qui constitue un élément indiscutable dans cette pathologie. L’acquisition d’une résistance endocrinienne est cependant un obstacle majeur au traitement de cette forme de cancer. L’émergence de cancers hormono-indépendants peut est produite par l’activation de ER en absence d’estrogène, l’hypersensibilité du récepteur aux faibles concentrations plasmique d’estrogène ainsi que l’activation de ER par des modulateurs sélectifs. L’activité du ER est fortement influencée par l’environnement cellulaire tel que l’activation de voie de signalisation des facteurs de croissances, la disponibilité de protéines co-régulatrices et des séquences promotrices ciblées. Présentement, les études ont principalement considérées le rôle de ERα, cependant avec la découverte de ERβ, notre compréhension de la diversité des mécanismes potentiels impliquant des réponses ER-dépendantes s’est améliorée. L’activation des voies des kinases par les facteurs de croissance entraîne le développement d’un phénotype tumoral résistant aux traitements actuels. Nos connaissances des voies impliquées dans l’activation de ER sont restreintes. ERα est considéré comme le sous-type dominant et corrèle avec la plupart des facteurs de pronostic dans le cancer du sein. Le rôle de ERβ reste imprécis. Les résultats présentés dans cette thèse ont pour objectif de mieux comprendre l’implication de ERβ dans la prolifération cellulaire par l’étude du comportement de ERβ et ERα suite à l’activation des voies de signalisation par les facteurs de croissance. Nous démontrons que l’activation des récepteurs de surfaces de la famille ErbB, spécifiquement ErbB2/ErbB3, inhibe l’activité transcriptionnelle de ERβ, malgré la présence du coactivateur CBP, tout en activant ERα. De plus, l’inhibition de ERβ est attribuée à un résidu sérine (Ser-255) situé dans la région charnière, absente dans ERα. Des études supplémentaires de ErbB2/ErbB3 ont révélé qu’ils activent la voie PI3K/Akt ciblant à son tour la Ser-255. En effet, cette phosphorylation de ERβ par PI3K/Akt induit une augmentation de l’ubiquitination du récepteur qui promeut sa dégradation par le système ubiquitine-protéasome. Cette dégradation est spécifique pour ERβ. De façon intéressante, la dégradation par le protéasome requiert la présence du coactivateur CBP normalement requis pour l’activité transcriptionnelle des récepteurs nucléaires. Malgré le fait que l’activation de la voie PI3K/Akt corrèle avec une diminution de l’expression des gènes sous le contrôle de ERβ, on observe une augmentation de la prolifération des cellules cancéreuses. L’inhibition de la dégradation de ERβ réduit cette prolifération excessive causée par le traitement avec Hrgβ1, un ligand de ErbB3. Un nombre croissant d’évidences indique que les voies de signalisations des facteurs de croissance peuvent sélectivement réguler l’activité transcriptionnelle de sous-types de ER. De plus, le ratio ERα/ERβ dans les cancers du sein devient un outil de diagnostique populaire afin de déterminer la sévérité d’une tumeur. En conclusion, la caractérisation moléculaire du couplage entre la signalisation des facteurs de croissance et la fonction des ERs permettra le développement de nouveaux traitements afin de limiter l’apparition de cellules tumorales résistantes aux thérapies endocriniennes actuelles. / It has long been appreciated that estrogenic signaling plays a critical role in the development of hormone-dependent cancers such as breast cancer. Two-thirds of breast cancers express estrogen receptor (ER) which has been demonstrated to play an irrefutable role in tumour development and progression. However the acquisition of endocrine resistance has become a major obstacle in the treatment of hormone-dependent cancers that have acquired a hormone-independent state. Hormone-independent cancers emerge from an array of pathways involving ER activation in the absence of estrogen, hypersensitivity of ER to low serum levels of estrogen and activation by estrogen antagonists. The activity of ER is critically influenced by the cellular environment such as growth factor signaling pathways, availability of coregulatory proteins and the promoter sequence of target genes. The mechanisms studied have mostly considered the role of ERα, however with the discovery of the second subtype, ERβ, the understanding on the diversity of potential mechanisms involving ER-dependent responses have improved. Hormonal-independent activation of ER can occur in estrogen-dependent breast tumours, with concomitant rise in kinase signaling pathways, resulting in the acquisition of a therapeutic resistant phenotype in treated women. Our knowledge is relatively limited on which pathways trigger ER signaling and how these phosphorylation-coupled events affect ER activity. ERα is considered the dominant subtype and correlates with most of the prognostic factors in breast cancers. Conversely the role of ERβ remains unclear. The results presented in this thesis were carried out with the objective of gaining a better understanding of ERβ’s role in cellular proliferation by examining the behavior of ERβ and ERα during the activation of growth factor signaling pathways by cell-surface receptor-tyrosine kinases. We demonstrate here that the activation of cell surface receptors of the ErbB family, specifically ErbB2/ErbB3, inhibits the transcriptional activity of ERβ despite the presence of the coactivator CBP, yet activated ERα. Furthermore the inhibition of ERβ was attributed to a specific serine residue located within the hinge region, not present in ERα. Additional studies of ErbB2/ErbB3-initiated signaling revealed that it triggered the activation of the PI3K/Akt pathway which targeted the serine residue within the hinge region of ERβ. In fact, phosphorylation of ERβ by the PI3K/Akt pathway led to an increase in receptor ubiquitination which promoted its degradation by the ubiquitin-proteasome system which was subtype specific. Interestingly, proteasomal degradation required the presence of the coactivator CBP, which is normally involved in assisting nuclear receptor transcriptional activity. Although the activation of the PI3K/Akt pathway correlated with a decrease in the expression of ERβ target genes it led to an increase in the proliferation of breast cancer cells. Inhibiting the degradation of ERβ reduced the enhanced proliferation of breast cancer cells brought about by the treatment of ErbB3’s ligand, Hrgβ1. Increasing evidence indicates that growth factor signaling pathways can selectively regulate the transcriptional activity of ER subtypes, and the ratio of ERα/ERβ expression in breast tumours is becoming a popular prognostic factor to evaluate the severity of the tumour. Therefore the molecular characterization of the coupling between growth factor signaling and ER function should provide improved therapeutical approaches to overcome or delay the onset of resistance to endocrine therapy in hormone-dependent cancers.

As proteínas p53, MDM-2, c-Kit, ErbB-2, PCNA e p-glicoproteína foram estudadas em 42 amostras de osteossarcoma ao diagnóstico, através da técnica de imunohistoquímica, e foram correlacionados estes achados com o prognóstico destes pacientes. O p-53 foi positivo em 23,1% (9/39), PCNA em 71,4% (25/35), p-glicoproteína em 40,5% (15/37), MDM-2 em 34,8% (8/23), c-kit em 67,6% (25/37) e ErbB-2 em 17,9% (7/39). Na análise univariada, a presença de metástases ao diagnóstico, a positividade de p53 e ErbB-2 influenciaram o prognóstico individualmente, entretanto, na análise multivariada, a presença de metástase ao diagnóstico revelou-se o único fator de prognóstico estatisticamente significante / We study, by imunohistochemistry technique, the proteins p53, MDM-2, c-Kit, ErbB-2, PCNA and p-glycoprotein in samples of osteosarcoma tumors at diagnosis and its correlation with the prognosis of this patients. The p-53 was positive in 23,1% (9/39), PCNA in 71,4% (25/35), p-glycoprotein in 40,5% (15/37), MDM-2 in 34,8% (8/23), c-kit in 67,8%(25/37) and ErbB-2 in 17,9% (7/39) of the samples. In the univariate analysis, the presence of metastasis at diagnosis, the positivity of p-53 and ErbB-2 influenced the prognosis individually, otherwise, in the multivariate analysis, the presence of metastasis at diagnosis was the only prgnostic factor statistically significant

Le cancer colorectal (CCR) est la troisième maladie maligne la plus fréquente et la deuxième cause de décès par cancer. La famille des récepteurs tyrosine kinases transmembranaires ErbB a été identifiée comme l'un des principaux moteurs du développement et de la progression du CCR et l'un de ses membres les plus connus, le récepteur du facteur de croissance épidermique (EGFR / ERBB1 / Her1), considéré comme l'une des cibles les plus importantes en traitement CRC. Deux autres membres de la famille ErbB, les récepteurs Her2 et Her3, apparaissent également comme de nouvelles cibles importantes pour le CRC en raison de la mutation somatique, de l’amplification génique ou de la résistance aux traitements anti-EGFR. La protéine transmembranaire, Fas (TNFRSF6 / CD95), est un membre de la superfamille des récepteurs du facteur de nécrose tumorale (TNFRSF). Il peut transmettre des signaux multiples qui mènent à des destins de cellules complètement différents. Selon les contextes cellulaires, Fas initie la mort cellulaire par apoptose, essentielle pour arrêter les réponses immunitaires chroniques et prévenir l'auto-immunité et le cancer, ou pour stimuler la survie, la prolifération et la motilité des cellules, ce qui favorise l'auto-immunité, la croissance cancéreuse et les métastases. Avec des preuves de plus en plus nombreuses de la signalisation pro-survie médiée par Fas, les activités de promotion du cancer chez les patients atteints de cancer sont maintenant reconnues comme étant significatives et cliniquement pertinentes. Bien que cette polyvalence de signalisation ait été particulièrement bien démontrée dans le cancer du côlon, les mécanismes moléculaires qui sous-tendent les voies de survie sont encore largement inconnus. Dans ce contexte, l'objectif principal de mon doctorat Le projet visait à étudier l’importance du crosstalks entre les membres de la famille Fas et ErbB et, plus particulièrement, à déterminer si la signalisation Fas pouvait influencer la signalisation de l’EGFR favorisant le cancer.Plus précisément, je décris comment l’état de phosphorylation de la tyrosine Fas influence fortement la signalisation de la voie EGFR dans les cellules colorectales. Mes données démontrent que Fas dans son état prosurvival, phosphorylé à Y291 (pY291-Fas), interagit en effet avec EGFR et que cette interaction intensifie significativement la signalisation de l'EGFR dans les cellules cancéreuses colorectales anti-EGFR via la voie Yes-1 / STAT3. Le pY291-Fas s'accumule dans le noyau lors du traitement par EGF et favorise la localisation nucléaire du phospho-EGFR et du phospho-STAT3, l'expression de la cycline D1, l'activation des voies Akt et MAPK médiées par STAT3 et enfin la prolifération et la migration cellulaires. De plus, je découvre également le rôle potentiel que Her3 pourrait jouer avec Fas dans la libération des cellules cancéreuses colorectales de l'inhibition anti-EGFR.Tous ensemble mon doctorat des études permet de mieux comprendre le rôle des voies de survie de Fas dans la signalisation ErBb dans le CRC. Fait important, en démontrant un lien entre l'émergence d'une résistance aux traitements anti-ErbB et le signal de Fas pro-survie, mon travail justifie le développement d'une thérapie ciblée Fas / phospho-Fas comme nouvelle option thérapeutique pour surmonter les anti-EGFR, chez les patients présentant une résistance anti-EGFR secondaire. / Colorectal cancer (CRC) is the third most common malignant disease and the second most frequent cause of cancer-related death. The ErbB family of transmembrane receptor tyrosine kinases has been identified as a major driver of the development and progression of CRC and one its best-known member, the epidermal growth factor receptor (EGFR /ERBB1/Her1), considered one of the most important targets in CRC treatment. Two others members of the ErbB family, the receptors Her2 and Her3, also emerge as important new targets for CRC due to the somatic mutation, gene amplification or resistance to the anti-EGFR therapies. The transmembrane protein, Fas (TNFRSF6/CD95), is a member of the tumor necrosis factor receptor superfamily (TNFRSF). It can transmit multiple signals that lead to completely different cell fates. Depending on cellular contexts, Fas either initiates cell death by apoptosis, which is essential for shutting down chronic immune responses and preventing autoimmunity and cancer, or stimulates cell survival, proliferation, and motility, which can promote autoimmunity, cancer growth, and metastasis. With increasing evidence of Fas-mediated pro-survival signaling, the cancer-promoting activities of Fas are now recognized as significant and clinically relevant. While this signaling versatility has been particularly well demonstrated in colon cancer, the molecular mechanisms underlying the survivals pathways are still largely unknown. In this context, the main aim of my Ph.D. project was to study the importance of the crosstalks between Fas and the ErbB family members and more specifically to determine whether the Fas signaling could influence the cancer-promoting signaling of EGFR.More precisely, I describe how the Fas tyrosine phosphorylation status strongly influences the signaling of the EGFR pathway in colorectal cells. My data demonstrate that Fas in its prosurvival state, phosphorylated at Y291 (pY291-Fas), indeed interacts with EGFR and that this interaction significantly intensifies EGFR signaling in anti-EGFR-resistant colorectal cancer cells via the Yes-1/STAT3-mediated pathway. The pY291-Fas accumulates in the nucleus upon EGF treatment and promotes the nuclear localization of phospho-EGFR and phospho-STAT3, the expression of cyclin D1, the activation of STAT3-mediated Akt and MAPK pathways, and finally the cell proliferation and migration. Additionally, I also uncover the potential role that Her3, may play along with Fas, in the colorectal cancer cell escape from anti-EGFR inhibition. All together my Ph.D. studies provide a better understanding of the role of the Fas survival pathways in the ErBb signaling in CRC. Importantly, by demonstrating a connection between the emergence of resistance to anti-ErbB therapies and the Fas pro-survival signal, my work provides a rationale for the development of Fas/phospho-Fas targeted therapy as a new therapeutic option for overcoming anti-EGFR, in patients with secondary anti-EGFR resistance.

Les acides biliaires sont cruciaux pour l’absorption intestinale des lipides et ils représentent une voie majeure d’élimination du cholestérol. À concentration élevée, ils sont cytotoxiques et potentiellement carcinogènes. Il est donc essentiel de maintenir des niveaux adéquats afin de préserver une homéostasie optimale. Le récepteur nucléaire FXR est grandement impliqué dans cette régulation, en étant activé par les acides biliaires qui agissent comme ligands et en régulant les gènes nécessaires à leur synthèse et leur métabolisme. FXR est aussi impliqué dans le métabolisme lipidique et glucidique, tout en ayant un rôle anti-inflammatoire et antiprolifératif. Les mécanismes régulant l’expression et l’activité transcriptionnelle de FXR sont toutefois peu connus. Leur caractérisation pourrait mener à l’identification de nouvelles cibles thérapeutiques pour les pathologies associées au syndrome métabolique. L’activation des récepteurs nucléaires peut se faire également de façon indépendante du ligand, soit via les modifications post-transcriptionnelles. Celles-ci permettent l’intégration d’une panoplie de signaux extracellulaires et l’adaptation de la réponse transcriptionnelle des récepteurs nucléaires aux variations de conditions cellulaires. La SUMOylation et l’ubiquitination sont deux modifications pouvant affecter la localisation cellulaire des récepteurs, leur interaction avec des partenaires protéiques, l’affinité de liaison au ligand, à l’ADN, leur dimérisation, la dégradation de leurs cibles et l’arrêt de la transcription. Étant donné le rôle important des modifications post-traductionnelles des récepteurs nucléaires en réponse aux divers signaux cellulaires, nous nous sommes intéressés particulièrement à leur impact sur la dégradation et l’activité transcriptionnelle de FXR. Nos études nous ont permis d’identifier et de caractériser un nouveau site de SUMOylation de FXR, impliqué dans la régulation du récepteur. Le résidu lysine responsable de conjuguer la protéine SUMO est localisé dans un motif non-consensus de SUMOylation, prénommé pSuM, qui est sous le contrôle de la phosphorylation d’un résidu serine régulé par la kinase CK2. Nous avons également déterminé que la modification de FXR par SUMO-2 permet le recrutement de l’ubiquitine E3-ligase SUMO-dépendante RNF4, qui induit l’ubiquitination et la dégradation de FXR. Cette cascade de signalisation est nécessaire pour l’activation transcriptionnelle de FXR et pour la régulation de l’expression des gènes cibles. Elle permet de contrôler ses niveaux protéiques de façon très dynamique et d’assurer ainsi une homéostasie optimale. Dans la deuxième étude, nous identifions un nouveau signal régulant l’activité transcriptionnelle de FXR. Les récepteurs tyrosine kinase de la famille EGFR/ErbB sont connus pour activer plusieurs voies de signalisation favorisant la croissance et la prolifération cellulaire. Cependant, leur expression et activité sont souvent altérées dans différents cancers, menant à une prolifération tumorale soutenue et dérégulée. Nous démontrons que l’activation des récepteurs de la famille EGFR/ErbB mène à la répression de l’activité transcriptionnelle de FXR en induisant la SUMOylation de FXR sur des résidus lysines situés dans des sites consensus de FXR. Étant donné le rôle antiprolifératif de FXR, l’impact répresseur des récepteurs ErbB sur l’activité de FXR pourrait contribuer à leur potentiel tumorigénique. Nos résultats approfondissent notre compréhension des mécanismes de régulation de l’expression et de l’activité de FXR. Étant donné son rôle important dans le métabolisme énergétique, la réponse transcriptionnelle de FXR doit être adaptée efficacement aux variations des conditions cellulaires dans un processus de régulation homéostatique. Les modifications post-traductionnelles assurent une régulation dynamique de l’activité de FXR et leur dérégulation pourrait être impliquée dans les pathologies associées au syndrome métabolique. / Bile acids are crucial for the absorption of intestinal lipids, and are directly involved in the efflux pathway to eliminate cholesterol. At high concentrations, bile acids are cytotoxic and potentially carcinogenic. It is therefore essential to maintain bile acids to adequate levels in order to preserve optimal homeostasis. Nuclear receptor FXR is directly involved in bile acid homeostasis by being activated by bile acids to regulate critical genes required for their synthesis and their metabolism. FXR is also involved in lipid and glucose metabolism, as well as having anti-inflammatory and anti-proliferative roles. However, the exact mechanisms regulating the degradation and activity of FXR are not well understood. Therefore, elucidation of FXR activity and response to cellular signals is essential to develop novel strategies and therapeutic targets for pathologies associated with the metabolic syndrome. Besides ligand activation, nuclear receptor can be regulated in a ligand-independent manner, mainly via post-translational modifications. Such modifications are important to allow homeostatic integration of diverse extracellular signals to ensure adaptation and transcriptional response of nuclear receptors. Among them, SUMOylation and ubiquitination are two modifications that modulate cellular localization of receptors, their interaction with protein partners, ligand binding and sensitivity, DNA affinity, receptor dimerisation, stability of their targets and transcriptional dynamics. Because of the important role of post-translational modifications in nuclear receptor function, we therefore study their specific impact in respect to FXR regulation and transcriptional competence. In this study, we have identified and characterized a new and non-consensus SUMOylation site involved in the regulation of FXR activity. This site, termed pSuM for phosphorylation-dependent SUMOylation motif, consists of a targeted lysine residue that conjugates SUMO proteins under the control of kinase CK2-mediated phosphorylation. We also determined that such modification of FXR with SUMO-2 induced the recruitment of SUMO-dependent E3 ligase RNF4, resulting in FXR ubiquitination and degradation. We demonstrate that this signaling cascade involving CK2 and RNF4 is required for FXR transcriptional activation and regulation of target gene expression. Our findings identify a cellular pathway that allows a dynamic control of FXR function to ensure efficient bile acid and energy metabolism in cells. In the second study, we identify a novel cellular signal that regulates FXR activity. Tyrosine kinase receptors of the EGFR/ErbB family are well known to participate in many signaling pathways, promoting cell growth and proliferation. Aberrant expression and activity of ErbB receptors are often associated to various cancers, leading to deregulated proliferation of tumors. Here, we show that ErbB activation leads to repression of FXR transcriptional activity by inducing FXR phosphorylation and specific SUMOylation at consensus sites. Because of the antiproliferative role of FXR, the negative impact of ErbB receptors on FXR transcriptional activity is thought to contribute to their tumorigenic potential. Altogether, our results expand our understanding of the mechanisms regulating FXR expression and activity. Because of its important role in lipid and energy metabolism, the transcriptional response of FXR needs to be efficiently adapted to variations of cellular conditions in order to achieve essential homeostatic control. As such, post-translational modifications ensure a dynamic regulation of FXR activity and their pathologic deregulation may be involved in diverse diseases associated with metabolic syndrome.

INTRODUÇÃO: Os astrocitomas difusamente infiltrativos são os tumores mais freqüentes de Sistema Nervoso Central (SNC) com uma taxa de 5-7 novos casos por 100.000 pessoas ano. São tumores altamente invasivos e estão associados com alterações de alguns genes como EGF (fator de crescimento epidérmico) e o EGFR (receptor do fator de crescimento epidérmico), que podem criar um aumento da atividade mitogênica, acarretando aumento de proliferação e maturação celular, apoptose, angiogênese e metástase. O nível de expressão destes genes pode ser influenciado por alterações genéticas, como a presença de polimorfismos. Uma mudança única de base (SNP) pode alterar a expressão gênica e, sendo assim, estar associada ao aumento do risco de desenvolver astrocitomas. Nesse trabalho, foram analisados 2 SNPs na região não traduzida (c.-191C>A e c.-216G>T) e um SNP no exon 16 (c.2073A>T) do gene EGFR, e um outro SNP na região não traduzida no gene EGF (c.61A>G). Os SNPs foram associados a expressão gênica do EGFR e a sobrevida dos pacientes. MÈTODOS: Foi realizado um estudo caso-controle com 193 casos de astrocitomas difusamente infiltrativos e 200 controles por amplificação por PCR seguido de digestão enzimática. Os produtos digeridos das amostras foram analisados por eletroforese em gel de agarose e poliacrilamida e corados com brometo de etídeo. A expressão gênica foi realizada após extração de RNA do tecido tumoral seguida de transcrição reversa e PCR em tempo real. Testes de qui-quadrado, odds ratio (OR), intervalo de confiança 95% (IC95%), t de Student e curvas de Kaplan-Meier foram realizados para análises estatística. RESULTADOS: A análise das freqüências dos genótipos dos polimorfismos mostrou uma diferença na distribuição entre casos e controles para o polimorfismo c.2073A>T. Pacientes com o genótipo TT apresentou um menor risco para astrocitoma quando comparados com o genótipo AA (OR=0,51, IC95%=0,29-0,99). Nenhuma correlação foi encontrada para os outros polimorfismos analisados. Também não foi encontrada correlação entre os genótipos dos polimorfismos e os níveis de expressão de EGFR e a sobrevida dos pacientes. CONCLUSÃO: Nosso trabalho mostrou haver um possível fator de proteção quando o paciente é portador do genótipo TT, o que pode levar a uma diminuição do risco de desenvolver o tumor. Pacientes com genótipo TT do polimorfismo c.2073A>T do gene EGFR apresentam um menor risco para astrocitomas difusamente infiltrativos do que os com o genótipo AA. / INTRODUCTION: Diffusely infiltrative astrocytomas are the most frequent tumors of the Central Nervous System (CNS) with a rate of 5-7 new cases in 100,000 individuals per year. They are highly invasive, and they are associated to alterations in some genes as EGF (epidermal growth factor) and EGFR (epidermal growth factor receptor), which may increase mitogenic activity, leading to increase of proliferation, cellular maturation, apoptosis, angiogenesis, and metastasis. Genetic alterations, as presence of polymorphisms of single nucleotide change (SNP) could influence their expression level, and thus could be associated to increased risk in developing astrocytomas. In the present study, two SNP of non-coding region (c.-191C>A and c.-216G>T) and one SNP in exon 16 (c.2073A>T) of EGFR, and another SNP of non-coding region of EGF (c.61A>G) were analyzed. The SNPs were associated to EGFR expression level and to survival time. METHOD: a case-control study of 193 of diffusely infiltrative astrocytomas and 200 controls was carried out, with PCR amplification and enzymatic digestion, which products were analyzed in agarose gel or polyacrylamide gel electrophoresis stained by ethidium bromide. EGFR expression level was studied by real time PCR after RNA extraction followed by reverse transcription of tumor tissues compared to epileptic non-neoplastic brain tissues. Stastistical analysis were performed by chi-square, odds ratio (OR), 95% confidence interval (95% CI), Student-t test and Kaplan Meier. RESULTS: The polymorphic genotype frequency was different between case and controls for the polymorphism c.2073A>T. Patients with TT genotype presented lower risk to develop astrocytoma when compared to genotype AA (OR=0.51, CI95%=0.29- 0.99). No other correlation was observed for the remaining studied polymorphisms. There was neither correlation between the polymorphic genotypes and the EGFR expression levels nor with survival time. CONCLUSION: The present study showed a possible protection factor in developing astrocytomas for the patients harboring the genotype TT of c.2073A>T polymorphism of EFGR, thus the patients presenting TT genotype have lower risk to develop diffusely infiltrative astrocytoma than patients presenting the genotype AA.

Introdução: O carcinoma de células renais (CCR) representa uma causa crescente de mortalidade por câncer. Apesar da sua curabilidade em estádios iniciais, agentes citotóxicos e imunomodulatórios têm homogeneamente alcançado nenhum ou pouco benefício no tratamento do CCR avançado. Um melhor entendimento da biologia tumoral pode levar ao desenvolvimento de terapias mais eficientes e dirigidas aos mecanismos moleculares de manutenção do tumor. Sinalização aumentada através do receptor do fator de crescimento epitelial (EGFR), sistema de quinases proteicas ativadas por mitógenos (MAPK) e via da fosfatidilinositol 3-quinase (PI3K) assim como estimulação do complexo de início de tradução (CIT) foram caracterizados em linhagens celulares e amostras tumorais de CCR. Nós investigamos o efeito de um inibidor de EGFR (gefitinibe), de um inibidor de MAPK (UO126) e de um inibidor do CIT (rapamicina) nos intermediários de sinalização celular e nos elementos do CIT assim como no crescimento in vitro de linhagens celulares de CCR. Métodos: Linhagens celulares de CCR (PRC3, WT8, SKRC-02, SKRC-17, SKRC-39, SKRC-45, ACHN e KRCY) foram mantidas em condições ideais para a cultura de células de mamíferos e expostas a drogas e/ou inibidores nas concentrações e por períodos de tempo variáveis. A fosforilação de intermediários da sinalização celular foi determinada utilizando-se western blots. Nível de mRNA para genes de interesse foram determinados por qRT-PCR. Crescimento celular foi avaliado por método colorimétrico em diferentes concentrações de gefitinibe, UO126 e rapamicina, isoladamente ou em combinação. Resultados: UO126 levou a defosforilação não apenas de intermediários de MAPK como também de substratos do alvo da rapamicina em mamíferos (mTOR) revelando sinalização cruzada entre essas vias. Nós identificamos ainda que ERK5 é a quinase potencialmente responsável por esta sinalização cruzada. No entanto, tratamento com UO126 não afetou o nível de mRNA para o substrato de mTOR 4EBP1. Gefitinibe foi capaz de bloquear a sinalização iniciada por EGF na via de PI3K em todas as linhagens wt-PTEN e de bloquear a sinalização através de ERK1/2 e ERK5 ao menos parcialmente em todas as linhagens celulares. Rapamicina mostrou-se um potente inibidor do crescimento na maioria das linhagens celulares de CCR e tal efeito foi freqüentemente amplificado com a combinação com UO126 ou gefitinibe.Conclusão: EGFR, MAPK e CIT são alvos promissores no tratamento do CCR / Background: Renal cell carcinoma (RCC) is an increasing cause of cancer mortality. Despite its curability in early stages, conventional cytotoxic and immunomodulatory agents have been homogeneous in providing minimal or no benefit in the treatment of advanced RCC. Better understanding of tumor cell biology may lead to development of more efficient targeted therapies. Signaling intensification through epithelial growth factor receptor (EGFR), mitogenactivated protein kinase (MAPK) pathway, Phosphatidylinositol 3-kinase (PI3K) pathway and overactivation of the translation initiation complex (TIC) has been previously characterized in RCC cell lines and tumor samples. We investigated the effect of an EGFR inhibitor (gefitinib) as well as a MAPK inhibitor (UO126) and a TIC inhibitor (rapamycin) in the intermediates of cell signaling, in the elements of TIC and in the in vitro growth of RCC cell lines. Methods: RCC cell lines (PRC3, WT8, SKRC-02, SKRC-17, SKRC-39, SKRC-45, ACHN and KRCY) were maintained on standard mammalian cells culture conditions and exposed to drugs and/or inhibitors in variable concentrations and for variable periods of time as required in each experiment. Phosphorylation status of signaling intermediates were determined using western blots. Levels of mRNA for genes of interest were determined by qRT-PCR. Cell growth was assessed by colorimetric method in control conditions or in different concentrations of gefitinib, UO126 or rapamycin, alone or in combination. Results: UO126 caused dephosphorylation not only of MAPK intermediates but also of mammalian target of rapamycin (mTOR) substrates revealing crosstalk between these pathways. We also identified ERK5 as a kinase potentially responsible for such cross talk. However, treatment with UO126 did not affect mRNA levels of the downstream target of mTOR 4EBP1. Gefitinib was able to block EGF signaling through PI3K in all wt- PTEN cell lines and the signaling through ERK1/2 and ERK5 at least partially in all cell lines. Rapamycin was found to be a potent growth inhibitor in most RCC cell lines and such effect was often increased by its combination with UO126 or gefitinib. Conclusion: EGFR, MAPK and CIT are promising targets for the treatment of RCC

Introdução: O carcinoma de células renais (CCR) representa uma causa crescente de mortalidade por câncer. Apesar da sua curabilidade em estádios iniciais, agentes citotóxicos e imunomodulatórios têm homogeneamente alcançado nenhum ou pouco benefício no tratamento do CCR avançado. Um melhor entendimento da biologia tumoral pode levar ao desenvolvimento de terapias mais eficientes e dirigidas aos mecanismos moleculares de manutenção do tumor. Sinalização aumentada através do receptor do fator de crescimento epitelial (EGFR), sistema de quinases proteicas ativadas por mitógenos (MAPK) e via da fosfatidilinositol 3-quinase (PI3K) assim como estimulação do complexo de início de tradução (CIT) foram caracterizados em linhagens celulares e amostras tumorais de CCR. Nós investigamos o efeito de um inibidor de EGFR (gefitinibe), de um inibidor de MAPK (UO126) e de um inibidor do CIT (rapamicina) nos intermediários de sinalização celular e nos elementos do CIT assim como no crescimento in vitro de linhagens celulares de CCR. Métodos: Linhagens celulares de CCR (PRC3, WT8, SKRC-02, SKRC-17, SKRC-39, SKRC-45, ACHN e KRCY) foram mantidas em condições ideais para a cultura de células de mamíferos e expostas a drogas e/ou inibidores nas concentrações e por períodos de tempo variáveis. A fosforilação de intermediários da sinalização celular foi determinada utilizando-se western blots. Nível de mRNA para genes de interesse foram determinados por qRT-PCR. Crescimento celular foi avaliado por método colorimétrico em diferentes concentrações de gefitinibe, UO126 e rapamicina, isoladamente ou em combinação. Resultados: UO126 levou a defosforilação não apenas de intermediários de MAPK como também de substratos do alvo da rapamicina em mamíferos (mTOR) revelando sinalização cruzada entre essas vias. Nós identificamos ainda que ERK5 é a quinase potencialmente responsável por esta sinalização cruzada. No entanto, tratamento com UO126 não afetou o nível de mRNA para o substrato de mTOR 4EBP1. Gefitinibe foi capaz de bloquear a sinalização iniciada por EGF na via de PI3K em todas as linhagens wt-PTEN e de bloquear a sinalização através de ERK1/2 e ERK5 ao menos parcialmente em todas as linhagens celulares. Rapamicina mostrou-se um potente inibidor do crescimento na maioria das linhagens celulares de CCR e tal efeito foi freqüentemente amplificado com a combinação com UO126 ou gefitinibe.Conclusão: EGFR, MAPK e CIT são alvos promissores no tratamento do CCR / Background: Renal cell carcinoma (RCC) is an increasing cause of cancer mortality. Despite its curability in early stages, conventional cytotoxic and immunomodulatory agents have been homogeneous in providing minimal or no benefit in the treatment of advanced RCC. Better understanding of tumor cell biology may lead to development of more efficient targeted therapies. Signaling intensification through epithelial growth factor receptor (EGFR), mitogenactivated protein kinase (MAPK) pathway, Phosphatidylinositol 3-kinase (PI3K) pathway and overactivation of the translation initiation complex (TIC) has been previously characterized in RCC cell lines and tumor samples. We investigated the effect of an EGFR inhibitor (gefitinib) as well as a MAPK inhibitor (UO126) and a TIC inhibitor (rapamycin) in the intermediates of cell signaling, in the elements of TIC and in the in vitro growth of RCC cell lines. Methods: RCC cell lines (PRC3, WT8, SKRC-02, SKRC-17, SKRC-39, SKRC-45, ACHN and KRCY) were maintained on standard mammalian cells culture conditions and exposed to drugs and/or inhibitors in variable concentrations and for variable periods of time as required in each experiment. Phosphorylation status of signaling intermediates were determined using western blots. Levels of mRNA for genes of interest were determined by qRT-PCR. Cell growth was assessed by colorimetric method in control conditions or in different concentrations of gefitinib, UO126 or rapamycin, alone or in combination. Results: UO126 caused dephosphorylation not only of MAPK intermediates but also of mammalian target of rapamycin (mTOR) substrates revealing crosstalk between these pathways. We also identified ERK5 as a kinase potentially responsible for such cross talk. However, treatment with UO126 did not affect mRNA levels of the downstream target of mTOR 4EBP1. Gefitinib was able to block EGF signaling through PI3K in all wt- PTEN cell lines and the signaling through ERK1/2 and ERK5 at least partially in all cell lines. Rapamycin was found to be a potent growth inhibitor in most RCC cell lines and such effect was often increased by its combination with UO126 or gefitinib. Conclusion: EGFR, MAPK and CIT are promising targets for the treatment of RCC

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.