Author Manuscript Published OnlineFirst on February 16, 2016; DOI: 10.1158/1940-6207.CAPR-15-0349 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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

7KHVHWZRDXWKRUVFRQWULEXWHGHTXDOO\WRWKLVVWXG\  &RQIOLFWVRILQWHUHVW7KHDXWKRUVGLVFORVHQRFRQIOLFWV 



ͳ

Downloaded from cancerpreventionresearch.aacrjournals.org on February 21, 2016. © 2016 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on February 16, 2016; DOI: 10.1158/1940-6207.CAPR-15-0349 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

$EVWUDFW ,QWHVWLQDOFDQFHULVDKHWHURJHQHRXVGLVHDVHGULYHQE\JHQHWLFPXWDWLRQVDQGHSLJHQHWLFFKDQJHV $SSUR[LPDWHO\  RI VSRUDGLF FRORUHFWDO FDQFHUV DUH LQLWLDWHG E\ PXWDWLRQ DQG LQDFWLYDWLRQ RI WKH adenomatous polyposis coli APC  JHQH ZKLFK UHVXOWV LQ XQUHVWUDLQHG LQWHVWLQDO HSLWKHOLDO JURZWK DQG IRUPDWLRQ RI DGHQRPDV $EHUUDQW '1$ PHWK\ODWLRQ SURPRWHV FDQFHU SURJUHVVLRQ E\ WKH LQDFWLYDWLRQ RI WXPRU VXSSUHVVRU JHQHV YLD SURPRWHU PHWK\ODWLRQ $GGLWLRQDOO\ JOREDO '1$ K\SRPHWK\ODWLRQ LV RIWHQ VHHQEHIRUHWKHIRUPDWLRQRIDGHQRPDVVXJJHVWLQJLWFRQWULEXWHVWRQHRSODVWLFWUDQVIRUPDWLRQ3UHYLRXV VWXGLHV HPSOR\HG PLFH ZLWK D K\SRPRUSKLF PXWDWLRQ LQ DNA methyltransferase 1 Dnmt1  ZKLFK H[KLELWHG FRQVWLWXWLYH JOREDO '1$ K\SRPHWK\ODWLRQ DQG GHFUHDVHG WXPRULJHQHVLV LQ WKH ApcMin/+ PRXVH PRGHO RI LQWHVWLQDO FDQFHU +RZHYHU WKH FRQVHTXHQFHV RI LQWHVWLQDOHSLWKHOLDO VSHFLILF DFXWH K\SRPHWK\ODWLRQGXULQJApcMin/+WXPRULQLWLDWLRQKDYHQRWEHHQUHSRUWHG 8VLQJWHPSRUDOO\FRQWUROOHGLQWHVWLQDOHSLWKHOLDOVSHFLILFJHQHDEODWLRQZHVKRZWKDWWRWDOORVVRI Dnmt1LQWKHApcMin/+PRXVHPRGHORILQWHVWLQDOFDQFHUFDXVHVDFFHOHUDWHGDGHQRPDLQLWLDWLRQ'HOHWLRQRI Dnmt1 SUHFLSLWDWHV DQ DFXWH UHVSRQVH FKDUDFWHUL]HG E\ K\SRPHWK\ODWLRQ RI UHSHWLWLYH HOHPHQWV DQG JHQRPLF LQVWDELOLW\ ZKLFK VXUSULVLQJO\ LV IROORZHG E\ UHPHWK\ODWLRQ ZLWK WLPH 7ZR PRQWKV SRVWDnmt1 DEODWLRQ PLFH GLVSOD\ LQFUHDVHG PDFURDGHQRPD ORDG FRQVLVWHQW ZLWK D UROH IRU 'QPW DQG '1$ PHWK\ODWLRQ LQ PDLQWDLQLQJ JHQRPLF VWDELOLW\ 7KHVH GDWD VXJJHVW WKDW '1$ K\SRPHWK\ODWLRQ SOD\V D SUHYLRXVO\XQDSSUHFLDWHGUROHLQLQWHVWLQDODGHQRPDLQLWLDWLRQ



ʹ

Downloaded from cancerpreventionresearch.aacrjournals.org on February 21, 2016. © 2016 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on February 16, 2016; DOI: 10.1158/1940-6207.CAPR-15-0349 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

,QWURGXFWLRQ 

,QWHVWLQDO DQG FRORUHFWDO FDQFHUV DUH PXOWLIDFWRULDO GLVHDVHV ZLWK YDULRXV LQSXWV LQFOXGLQJ GLHW

HQYLURQPHQW JHQHWLF PXWDWLRQV DQG HSLJHQHWLF DEQRUPDOLWLHV 7KH GLVHDVH ILUVW PDQLIHVWV DV DQ RYHU SUROLIHUDWLRQGHIHFWLQWKHIRUPRISRO\SVWKDWLIQRWUHPRYHGFDQSURJUHVVWRSUHFDQFHURXVDGHQRPDV )XUWKHU WUDQVLWLRQ WR LQYDVLYH DQG PHWDVWDWLF FDQFHU LV GXH WR WKH DFFXPXODWLRQ RI PXOWLSOH JHQHWLF PXWDWLRQVDQGHSLJHQHWLFFKDQJHVWKDWDOWHUJHQHH[SUHVVLRQSDWWHUQVGULYLQJQHRSODVWLFWUDQVIRUPDWLRQ DQGJURZWK  +RZHYHUWKHH[WHQWWRZKLFKHSLJHQHWLFPRGLILFDWLRQVDQGVSHFLILFDOO\'1$PHWK\ODWLRQ FRQWULEXWHWRWKHLQLWLDWLRQDQGSURJUHVVLRQRILQWHVWLQDOFDQFHULVXQFOHDU 1HRSODVWLF WLVVXHV IUHTXHQWO\ GLVSOD\ JOREDO '1$ K\SRPHWK\ODWLRQ ZKLFK FDQ RFFXU SDVVLYHO\ WKURXJK ORVV RI WKH PDLQWHQDQFH PHWK\OWUDQVIHUDVH '107 RU DFWLYHO\ E\ 7(7 HQ]\PHPHGLDWHG R[LGDWLRQ RI PHWK\OF\WRVLQH IROORZHG E\ EDVH H[FLVLRQ UHSDLU   $OWHUQDWLYHO\ VRPH WXPRUV H[KLELW LQFUHDVHG '1$ PHWK\ODWLRQ RQ VSHFLILF WXPRU VXSSUHVVRU JHQH SURPRWHUV ZKLFK UHTXLUHV WKH de novo '1$PHWK\OWUDQVIHUDVHV'107$DQG'107%+\SHUPHWK\ODWLRQRIWXPRUVXSSUHVVRUJHQHSURPRWHUV FDXVHV GHFUHDVHG DFWLYLW\ RI WKH DVVRFLDWHG JHQHV DQG SURPRWHV FDQFHU FHOO JURZWK in vitro   &RPSDULVRQRIKXPDQFRORQDGHQRPDVWRFRQWUROWLVVXHUHYHDOHGWKDWDOWKRXJKSUR[LPDOSURPRWHU&S* LVODQGV DUH RIWHQ K\SHUPHWK\ODWHG LQ WXPRUV '1$ K\SRPHWK\ODWLRQ LV IRXQG JHQRPHZLGH LQFOXGLQJ QHLJKERULQJ &S* LVODQG VKRUHV DQG UHSHWLWLYH HOHPHQWV   +\SRPHWK\ODWHG LQWHUJHQLF DQG LQWURQLF UHJLRQVDSSHDUHDUO\LQWKHWUDQVLWLRQIURPQRUPDOWRQHRSODVWLF  VXJJHVWLQJDPRUHLPSRUWDQWUROHIRU '1$K\SRPHWK\ODWLRQLQFDQFHULQLWLDWLRQWKDQSUHYLRXVO\DSSUHFLDWHG 3UHYLRXVO\WKHUROHRI'QPWLQLQWHVWLQDOWXPRULJHQHVLVZDVVWXGLHGLQWKHApcMin/+PRXVHPRGHO 7KLV SDUDGLJP PLPLFV WKH KHUHGLWDU\ KXPDQ FRORQ FDQFHU V\QGURPH )DPLOLDO $GHQRPDWRXV 3RO\SRVLV )$3 ZKLFKLVFDXVHGE\JHUPOLQHPXWDWLRQVRIWKHAPCJHQH  /RVVRIKHWHUR]\JRVLW\ /2+ DWWKH APC ORFXV FDXVHV ȕFDWHQLQ VWDELOL]DWLRQ DQG XQUHVWULFWHG :QW DFWLYLW\ UHVXOWLQJ LQ WKH IRUPDWLRQ RI PDFURVFRSLF DGHQRPDV E\  PRQWK RI DJH   /2+ LV DOVR D FRPPRQ WULJJHULQJ PHFKDQLVP IRU VSRUDGLFLQWHVWLQDODQGFRORUHFWDOWXPRUGHYHORSPHQW  UHQGHULQJWKHApcMin/+PRXVHDXVHIXOPRGHOIRU WKHVWXG\RILQWHVWLQDOFDQFHULQLWLDWLRQ 0XOWLSOH K\SRPRUSKLF Dnmt1 SDUDGLJPV VKRZ FRPSOHWH EORFN RI PDFURVFRSLF WXPRU IRUPDWLRQ  7KHDXWKRUVFRQFOXGHGWKDW'QPWDQG'1$PHWK\ODWLRQDUHUHTXLUHGIRUDGHQRPDGHYHORSPHQW



͵

Downloaded from cancerpreventionresearch.aacrjournals.org on February 21, 2016. © 2016 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on February 16, 2016; DOI: 10.1158/1940-6207.CAPR-15-0349 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

LQ WKH ApcMin/+ PRGHO 7KHVH VWXGLHV HPSOR\HG K\SRPRUSKLF Dnmt1 PLFH ZKLFK H[SUHVV FRQVWLWXWLYHO\ UHGXFHG'QPWOHYHOVIURPHDUOLHVWGHYHORSPHQWRQZDUGLQDOOWLVVXHV  LQFOXGLQJQRQHSLWKHOLDOFHOOV VXFKDVP\RILEUREODVWV0HVHQFK\PDOO\H[SUHVVHGJHQHVKDYHEHHQVKRZQWREHVWURQJPRGLILHUVRIWKH ApcMin/+WXPRUORDG  DQGFRXOGWKXVFRQWULEXWHWRWKHREVHUYHGSKHQRW\SHLQWKHDnmt1K\SRPRUSKLF ApcMin/+ LQWHVWLQH )XUWKHUPRUH FRQWLQXRXV '1$ K\SRPHWK\ODWLRQ PD\ PDVN DFXWH DIIHFWV GXULQJ WKH QHRSODVWLFWUDQVIRUPDWLRQZKLFKRFFXUVSULRUWRUHSRUWHGREVHUYDWLRQVRIPDFURDGHQRPDVDWVL[PRQWKV RIDJH'XHWRWKHVHOLPLWDWLRQVWKHSUHFLVHPHFKDQLVPRIKRZ'1$PHWK\ODWLRQDFWVZLWKLQWKHLQWHVWLQDO HSLWKHOLXPGXULQJDGHQRPDLQLWLDWLRQUHPDLQVWREHGHWHUPLQHG 7R DGGUHVV WKLV LPSRUWDQW NQRZOHGJH JDS ZH HPSOR\HG D WHPSRUDOO\ FRQWUROOHG LQWHVWLQDO HSLWKHOLDOVSHFLILF JHQH DEODWLRQ PRGHO WR GHOHWH Dnmt1 DQG GHFUHDVH PDLQWHQDQFH '1$ PHWK\ODWLRQ $EODWLRQ RI Dnmt1 FDXVHV DQ DFXWH SKHQRW\SH FKDUDFWHUL]HG E\ JOREDO '1$ K\SRPHWK\ODWLRQ JHQRPH LQVWDELOLW\DQGDSRSWRVLV7KHVHYHUHHIIHFWVRIDnmt1DEODWLRQUHVXOWLQDGUDPDWLFLQFUHDVHLQDGHQRPD LQLWLDWLRQE\DFFHOHUDWHGORVVRIKHWHUR]\JRVLW\DWWKHApcORFXV7KHVHUHVXOWVVWURQJO\VXSSRUWDUROHIRU '1$K\SRPHWK\ODWLRQLQFKURPRVRPDOLQVWDELOLW\DQGWXPRULQLWLDWLRQ



Ͷ

Downloaded from cancerpreventionresearch.aacrjournals.org on February 21, 2016. © 2016 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on February 16, 2016; DOI: 10.1158/1940-6207.CAPR-15-0349 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

0DWHULDOVDQG0HWKRGV  Mice. Dnmt1OR[3OR[3 PLFH ZHUH SURYLGHG E\ 5XGROI -DHQLVFK   9LOOLQ&UH(57 PLFH ZHUH UHFHLYHG IURP 6\OYLD 5RELQH   DQG ApcMin/+ PLFH IURP WKH -DFNVRQ /DERUDWRULHV   )RU Dnmt1 GHOHWLRQ H[SHULPHQWV &UHUHFRPELQDWLRQ DFWLYLW\ ZDV LQGXFHG E\ WKUHH GDLO\ LQWUDSHULWRQHDO LQMHFWLRQV RI  PJ WDPR[LIHQ 6LJPD LQDQHWKDQROVXQIORZHURLOPL[WXUH,QDOOH[SHULPHQWVOLWWHUPDWHFRQWUROVZLWKRXWWKH 9LOOLQ&UH(57 WUDQVJHQH ZHUH DOVR WDPR[LIHQ WUHDWHG $OO SURFHGXUHV LQYROYLQJ PLFH ZHUH FRQGXFWHG LQ DFFRUGDQFHZLWKDSSURYHG,QVWLWXWLRQDO$QLPDO&DUHDQG8VH&RPPLWWHHSURWRFROV Histology. 7LVVXHVZHUHLVRODWHGDQGIL[HGXVLQJSDUDIRUPDOGHK\GHLQ3%6DQGWKHQHPEHGGHGLQ SDUDIILQ $QWLJHQ UHWULHYDO ZDV SHUIRUPHG XVLQJ WKH  $QWLJHQ5HWULHYHU LQ %XIIHU $ (OHFWURQ 0LFURVFRS\6FLHQFHV DQGVWDQGDUGLPPXQRVWDLQLQJSURFHGXUHVZHUHSHUIRUPHGIRU'QPW 6DQWD&UX]  (&DGKHULQ %'7UDQVGXFWLRQ/DE 6R[ 0LOOLSRUH .L %'3KDUPLQJHQ ȕFDWHQLQ %'7UDQVGXFWLRQ /DE  &\F' %LRFDUH 0HGLFDO  (SFDP $EFDP  DQG γ+$; &HOO 6LJQDOLQJ  781(/ VWDLQLQJ ZDV SHUIRUPHGXVLQJ781(//DEHODQG(Q]\PH 5RFKH DQG$OH[D)OXRUDKDG873 /LIH7HFKQRORJLHV  3HUFHQWDJHV RI 781(/ QXFOHL ZHUH GHWHUPLQHG E\ FRXQWLQJ WKH QXPEHU RI QXFOHL SRVLWLYH IRU 781(/ VWDLQLQJ DQG GLYLGLQJ E\ WKH WRWDO QXPEHU RI QXFOHL LQ WKH LPDJH 7KH VDPH PHWKRG ZDV SHUIRUPHG WR TXDQWLI\WKHSHUFHQWDJHRIγ+$;FHOOV$OOPLFURVFRS\ZDVSHUIRUPHGRQD1LNRQ(FOLSVHL Laser Capture Microdissection. 7XPRUDQGQRQWXPRUHSLWKHOLDOFHOO'1$ZDVFROOHFWHGXVLQJD/HLFD /0' /DVHU 0LFURGLVVHFWLRQ PLFURVFRSH DQG WKH $UFWXUXV 3LFR3XUH '1$ LVRODWLRQ NLW $SSOLHG %LRV\VWHPV  Targeted Bisulfite Sequencing.  QJ RI PRXVH JHQRPLF '1$ ZDV ELVXOILWH FRQYHUWHG XVLQJ WKH (SLWHFWELVXOILWHNLW 4LDJHQ 7HPSODWH'1$ZDVDPSOLILHGXVLQJ.$3$+,),8UDFHO .$3$ ZLWKSULPHUV GLUHFWHGWRWKH/,1(HOHPHQWV  WKHH19LPSULQWLQJFRQWUROUHJLRQOlfm4DQGHes1  VdrHic1 Sfrp5MgmtDusp6c-FosMslnDQGS100a43ULPHUVHWVDQGUHJLRQVVXEVHTXHQWO\VHTXHQFHGFDQEH IRXQG LQ 6XSSOHPHQWDO 7DEOH  6HTXHQFLQJ OLEUDULHV ZHUH SUHSDUHG DQG DQDO\]HG XVLQJ WKH %L63&5 VWUDWHJ\ /,1( H19  RU WKH VWDQGDUG SURWRFRO Vdr, Hic1, Sfrp5, Mgmt, Dusp6, c-Fos, Msln, S100a4  GHVFULEHGSUHYLRXVO\   Tumor Evaluation. 0DFURVFRSLF WXPRUV ZHUH FRXQWHG LPPHGLDWHO\ DIWHU WLVVXH LVRODWLRQ XVLQJ D VWHUHRVFRSH$GHQRPDVL]HZDVFDOFXODWHGE\PHDVXULQJWKHZLGWKRIQHRSODVWLFOHVLRQVRQ+ (VWDLQHG



ͷ

Downloaded from cancerpreventionresearch.aacrjournals.org on February 21, 2016. © 2016 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on February 16, 2016; DOI: 10.1158/1940-6207.CAPR-15-0349 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

VPDOO LQWHVWLQH VHFWLRQV XVLQJ ,PDJH- +LVWRORJLFDO SDWKRORJ\ ZDV SHUIRUPHG E\ WKH &RPSDUDWLYH 3DWKRORJ\&RUHDWWKH8QLYHUVLW\RI3HQQV\OYDQLD6FKRRORI9HWHULQDU\0HGLFLQHIROORZLQJWKHJXLGHOLQHV DVGHVFULEHGSUHYLRXVO\  /2+ZDVGHWHUPLQHGDVGHVFULEHGSUHYLRXVO\   Phosphorylated H3 (pH3) cell cycle quantification. ApcMin/+;Dnmt1OR[3OR[39LOOLQ&UH(57PXWDQWVDQG VLEOLQJ ApcMin/+;Dnmt1OR[3OR[3 FRQWUROV ZHUH LQMHFWHG ZLWK WDPR[LIHQ DW  PRQWK RI DJH 6PDOO LQWHVWLQHV ZHUHFROOHFWHGZHHNPRQWKDQGPRQWKVIROORZLQJWDPR[LIHQDGPLQLVWUDWLRQLQVHSDUDWHFRKRUWVRI PLFH)ROORZLQJIL[DWLRQWLVVXHVHFWLRQVZHUHFRVWDLQHGIRUSKRVSKRU\ODWHGKLVWRQH+ 3+ WRLGHQWLI\ FHOOV LQ 0SKDVH DQG βFDWHQLQ WR LGHQWLI\ UHJLRQV RI K\SHUSUROLIHUDWLYH QHRSODVWLF HSLWKHOLXP :H FRXQWHGWKHQXPEHURI3+QXFOHLDQGGLYLGHGLWE\WKHWRWDOQXPEHURIQXFOHLLQWKHUHVSHFWLYHUHJLRQ 7KLV FDOFXODWLRQ ZDV DOVR SHUIRUPHG IRU QHRSODVWLF LQWHVWLQDO HSLWKHOLXP DW ERWK  PRQWK DQG  PRQWKV SRVWWDPR[LIHQWLPHSRLQWVIRUFRQWUROVDQGPXWDQWV q-RTPCR.,QWHVWLQHVLVRODWHGZHHNSRVWWDPR[LIHQZHUHJHQWO\VFUDSHGWRUHPRYHYLOOLDQGWUHDWHGZLWK ('7$ WR LVRODWH FU\SW FHOOV 0DFURVFRSLF WXPRUV DQG DGMDFHQW QRUPDO WLVVXH IURP LQWHVWLQHV LVRODWHG  PRQWKVSRVWWDPR[LIHQZHUHYLVXDOL]HGDQGGLVVHFWHGXVLQJDVWHUHRVFRSH51$ZDVH[WUDFWHGXVLQJWKH 7UL]RO51$LVRODWLRQSURWRFRO ,QYLWURJHQ IROORZHGE\51$FOHDQXSXVLQJWKH51HDV\0LQL.LW 4LDJHQ  P51$ H[SUHVVLRQ ZDV PHDVXUHG XVLQJ TXDQWLWDWLYH 573&5 DV GHVFULEHG SUHYLRXVO\   7KH 6

DNA Hypomethylation Contributes to Genomic Instability and Intestinal Cancer Initiation.

Intestinal cancer is a heterogeneous disease driven by genetic mutations and epigenetic changes. Approximately 80% of sporadic colorectal cancers are ...
2MB Sizes 0 Downloads 7 Views