You are on page 1of 36

Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.

CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.




PLFUR51$DS,QKLELWV&DQFHU&HOO0LJUDWLRQDQG$QJLRJHQHVLVYLD

'RZQUHJXODWLRQRI0DWUL[0HWDOORSURWHLQDVH  

<L\L/L&HP.XVFXφ$QQD%DQDFK4LDQ=KDQJ$VKOHLJK3XONRVNL*URVV

'HERUDK.LP-LQJ[XDQ/LX(ULF5RWK(OOHQ/L.HQQHWK56KUR\HU3DXOD,

'HQR\D;LDR[LD=KX♦/RQJKXD&KHQ♦DQG-LDQ&DR♦ 


'HSDUWPHQWRI0HGLFLQH&DQFHU3UHYHQWLRQ'HSDUWPHQWRI3DWKRORJ\'HSDUWPHQWRI

0HGLFLQH'LYLVLRQRI*DVWURHQWHURORJ\DQG+HSDWRORJ\'HSDUWPHQWRI6XUJHU\6WRQ\

%URRN8QLYHUVLW\6WRQ\%URRN1<86$'HSDUWPHQWRI5DGLDWLRQ2QFRORJ\

1DQIDQJ+RVSLWDO6RXWKHUQ0HGLFDO8QLYHUVLW\*XDQJ]KRX&KLQD 

φ
&XUUHQW$GGUHVV'HSDUWPHQWRI%LRFKHPLVWU\DQG0ROHFXODU*HQHWLFV8QLYHUVLW\RI

9LUJLQLD&KDUORWWHVYLOOH9$

 ♦&RUUHVSRQGHQFH]KX[[#FRPFKHQORQJKXDIO#FRPDQGMLDQFDR#VXQ\VEHGX

7KLV ZRUN LV VXSSRUWHG LQ SDUW E\ WKH %DOGZLQ %UHDVW &DQFHU )RXQGDWLRQ DQG WKH

1DWLRQDO&DQFHU,QVWLWXWH 5&$WR-& E\7KH6LPRQV)RXQGDWLRQ WR(/ E\

WKH 1DWLRQDO 1DWXUDO 6FLHQFH )RXQGDWLRQ RI &KLQD + WR /+&  DQGE\WKH

([FHOOHQW<RXQJ7HDFKHUV3URJUDPRI+LJKHU(GXFDWLRQRI*XDQJGRQJ3URYLQFH <T

WR;;= 

5XQQLQJWLWOH5HJXODWLRQRI003E\PL5DS

.H\ZRUGV PL5DS 003 0LJUDWLRQ ,QYDVLRQ $QJLRJHQHVLV DQG P51$


875 

7KHDXWKRUVKDYHQRFRQIOLFWRILQWHUHVWDVVRFLDWHGZLWKWKLVPDQXVFULSW 




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.




$EVWUDFW

 8SUHJXODWLRQRIPDWUL[PHWDOORSURWHLQDVH003 07003 LVDVVRFLDWHGZLWKSRRU

SURJQRVLVLQFDQFHUSDWLHQWVEXWLWLVXQFOHDUKRZ003EHFRPHVHOHYDWHGLQWXPRUV+HUH

ZHVKRZWKDW PL5DSLVGRZQUHJXODWHGLQDJJUHVVLYHKXPDQEUHDVWDQGFRORQFDQFHUV

ZKHUHLWVOHYHOVFRUUHODWHLQYHUVHO\ZLWK003H[SUHVVLRQ,QFOLQLFDOVSHFLPHQVHQKDQFHG

H[SUHVVLRQRI003ZDVREVHUYHGLQFDQFHUFHOOVORFDWHGDWWKHLQYDVLYHIURQWRIWXPRUV

ZKHUH PL5DS ZDV GRZQUHJXODWHG UHODWLYH WR DGMDFHQW QRUPDO FHOOV %LRLQIRUPDWLFV

DQDO\VHVGHILQHGDSRWHQWLDOPL5DSUHVSRQVHHOHPHQWZLWKLQWKH
XQWUDQVODWHGUHJLRQ

875  RI 003 WKDW ZDV YDOLGDWHG LQ UHSRUWHU JHQH H[SHULPHQWV (FWRSLF PL5DS

UHGXFHG 003 H[SUHVVLRQ ZKHUHDV PL5DS DWWHQXDWLRQ HOHYDWHG 003

H[SUHVVLRQ ,Q VXSSRUW RI D FULWLFDO UHODWLRQVKLS EHWZHHQ WKHVH WZR JHQHV

PL5DSPHGLDWHG UHGXFWLRQ RI 003 OHYHOV ZDV VXIILFLHQW WR GHFUHDVH FDQFHU FHOO

PLJUDWLRQLQYDVLRQDQGDFWLYDWLRQRISUR003)XUWKHUWKLVUHGXFWLRQLQ003OHYHOV

ZDVVXIILFLHQWWRUHGXFHLQYLYRLQYDVLRQDQGDQJLRJHQHVLVLQFKLFNFKRULRDOODQWRLFPHPEUDQH

DVVD\V 7DNHQ WRJHWKHU RXU UHVXOWV HVWDEOLVK WKH UHJXODWLRQ RI 003 LQ FDQFHUV E\

PL5DSWKURXJKDSRVWWUDQVFULSWLRQDOPHFKDQLVPDQGWKH\IXUWKHUVXJJHVWVWUDWHJLHVWR

HOHYDWHPL5DSWRSUHYHQWFDQFHUPHWDVWDVLV
 




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.




,QWURGXFWLRQ

0HWDVWDVLVWKHGLVVHPLQDWLRQRIFDQFHUFHOOVIURPWKHSULPDU\WXPRUWRDGLVWDQWRUJDQ

DFFRXQWV IRU  RI KXPDQ FDQFHUUHODWHG GHDWKV 3DWKRORJLFDOO\ PHWDVWDVLV LV D PXOWLVWHS

SURFHVVFRQVLVWLQJRIDVHULHVRIGLVFUHWHELRORJLFDOSURFHVVHV2QHRIWKHNH\ELRORJLFDOVWHSV

LVFDQFHUFHOOLQYDVLRQZKLFKLVLQYROYHGLQDOPRVWHYHU\VWDJHRIWKHPHWDVWDWLFFDVFDGHIURP

LQLWLDWLRQRIPHWDVWDVLVWRHVWDEOLVKPHQWRIDVHFRQGDU\WXPRU 

0DWUL[ PHWDOORSURWHLQDVHV 003V  DUH D IDPLO\ RI WZHQW\ILYH KLJKO\ KRPRORJRXV

=QGHSHQGHQW HQGRSHSWLGDVHV   7KH SULPDU\ IXQFWLRQV RI 003V DUH WUDGLWLRQDOO\

FRQVLGHUHG WR EH WKH GHJUDGDWLRQ RI FHOO DGKHVLRQ PROHFXOHV DQG UHPRYDO RI H[WUDFHOOXODU

PDWUL[ (&0 WRSHUPLWFDQFHUFHOOLQYDVLRQ  003VKDYHEHHQUHFHQWO\UHFRJQL]HGDVFHOO

PLJUDWLRQHQKDQFHUVLQGHSHQGHQWRIWKHLUSURWHRO\WLFDFWLYLW\  0DWUL[PHWDOORSURWHLQDVH

003 LVDPHPEUDQHLQVHUWHG003ZKLFKKDVEHHQIRXQGWRSOD\FULWLFDOUROHVLQFDQFHU

LQYDVLRQ DQG PHWDVWDVLV E\ FOHDYLQJ (&0 DQG EDVHPHQW PHPEUDQH SURWHLQV DFWLYDWLQJ

SUR003 DQG  LQGXFLQJ WKH DFWLYDWLRQ RI JURZWK IDFWRUV DQG HQKDQFLQJ FHOO

PLJUDWLRQ  3KHQRW\SLFDOO\003SOD\VDFULWLFDOUROHLQFRQYHUWLQJHSLWKHOLDOFHOOVWR

PLJUDWRU\ PHVHQFK\PDOOLNH FHOOV HSLWKHOLDOWRPHVHQFK\PDO WUDQVLWLRQ (07   ZKLFK LV

FRQVLGHUHG DQ LPSRUWDQW PHFKDQLVP IRU WKH LQLWLDO VWHS LQ WKH PHWDVWDWLF SURFHVV +RZHYHU

WKH UHJXODWRU\ PHFKDQLVP RI 003 H[SUHVVLRQ GXULQJ FDQFHU SURJUHVVLRQ UHPDLQV WR EH

FKDUDFWHUL]HG

,WKDVEHHQUHSRUWHGWKDW003H[SUHVVLRQLVXSUHJXODWHGE\WUDQVFULSWLRQIDFWRUVVXFK

DV6SDQG(JU  003KDVDOVREHHQGHPRQVWUDWHGWREHWUDQVFULSWLRQDOO\UHJXODWHG

E\ K\SR[LD LQGXFLEOH IDFWRUα    1RW RQO\ FDQ 003 H[SUHVVLRQ EH SRVLWLYHO\




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.




UHJXODWHG E\ WKHVH WUDQVFULSWLRQ IDFWRUV LW FDQ DOVR EH QHJDWLYHO\ UHJXODWHG E\ PLFUR51$

VXFKDVPLFUR51$DQGD   

PLFUR51$V PL51$VRUPL5 DUHVPDOOKLJKO\FRQVHUYHGQRQFRGLQJ51$VWKDWKDYH

EHHQ UHSRUWHG WR SDUWLFLSDWH LQ WKH PHWDVWDWLF SURFHVV E\ QHJDWLYHO\ RU SRVLWLYHO\ UHJXODWLQJ

JHQH H[SUHVVLRQ RI PHWDVWDVLVDVVRFLDWHG JHQHV WKURXJK SRVWWUDQVFULSWLRQDO UHSUHVVLRQ

P51$GHJUDGDWLRQRUSURPRWHUDFWLYDWLRQ  $OWKRXJKPL51$VFDQSRVLWLYHO\DIIHFWJHQH

H[SUHVVLRQ E\ ELQGLQJ WR WKH SURPRWHU UHJLRQ RI D UHJXODWHG JHQH WKH PDMRULW\ RIPL51$V

DUH UHSRUWHG WR QHJDWLYHO\ UHJXODWH WDUJHW JHQH H[SUHVVLRQ E\ UHSUHVVLQJ WUDQVODWLRQ RU

LQGXFLQJ VHTXHQFHVSHFLILF GHJUDGDWLRQ RI WDUJHW P51$V WKURXJK LQWHUDFWLRQ ZLWK WKH 


XQWUDQVODWHGUHJLRQV 
875V RIWDUJHWP51$V   

PL51$VDUHWUDQVFULEHGDVaQXFOHRWLGHSUHFXUVRUVDQGVXEVHTXHQWO\SURFHVVHGE\WKH

51DVH,,,W\SHHQ]\PH'LFHUWRJLYHDaQXFOHRWLGHPDWXUHSURGXFW  0RUHWKDQ

PL51$ JHQHV KDYH EHHQ LGHQWLILHG LQ WKH KXPDQ JHQRPH WKDW FROOHFWLYHO\ FRQWURO DQ

HVWLPDWHG  RI DOO KXPDQ JHQHV   (DFK PL51$ DSSHDUV WR UHJXODWH WKH H[SUHVVLRQ RI

WHQVWRKXQGUHGVRIJHQHVWRHIILFLHQWO\FRRUGLQDWHPXOWLSOHFHOOXODUSDWKZD\V,QWHUHVWLQJO\

PDQ\PL51$VH[LVWDVDPXOWLPHPEHUIDPLO\LQGLFDWLQJWKHLUIXQFWLRQDOUHGXQGDQF\ 

PLFUR51$DS PL5DS EHORQJVWRWKHPL5VIDPLO\ZKLFKLQFOXGHVIRXU

KLJKO\ FRQVHUYHG PDWXUH PL51$V PL5D E F DQG G 7KH\ DUH GHULYHG LQGHSHQGHQWO\

IURP VL[ SUHFXUVRUV ORFDWHG RQ WKUHH GLIIHUHQW FKURPRVRPHV  7KLV QHZ FODVV RI JHQHV KDV

UHFHQWO\ EHHQ VKRZQ WR SOD\ D FHQWUDO UROH LQ PDOLJQDQW WUDQVIRUPDWLRQ   ,Q FRQWUDVW

PL51$V DUH GRZQUHJXODWHG LQ PDQ\ WXPRUV DQG WKXV DSSHDU WR IXQFWLRQ DV WXPRU

VXSSUHVVRUJHQHV  




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.




,QWKLVVWXG\ZHLGHQWLILHGPL5DSDVDQHJDWLYHUHJXODWRURI003E\GLUHFWO\

WDUJHWLQJWKH
875RI003P51$UHVXOWLQJLQGHFUHDVHGFHOOPLJUDWLRQLQYDVLRQDQG

DQJLRJHQHVLV 2XU GDWD KLJKOLJKW D IXQFWLRQDO UROH RI PL5DS LQ FDQFHU GLVVHPLQDWLRQ

DQG XQFRYHU D SRWHQWLDO SURJQRVWLF ELRPDUNHU DQG PROHFXODU WDUJHW IRU WKH SUHYHQWLRQ RI

FDQFHUPHWDVWDVLV 

 




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.




0DWHULDOVDQG0HWKRGV

Cell Culture and Transfection:$OOFHOOOLQHVZHUHSXUFKDVHGIURP$7&& 0DQDVVDV9$ 

DQGZHUHPDLQWDLQHGLQ'0(0PHGLXPFRQWDLQLQJ)%6XQGHUD&2DWPRVSKHUH

+XPDQ EUHDVW FDQFHU VWHPOLNH 6.UG FHOOV ZHUH PDLQWDLQHG DV SUHYLRXVO\ GHVFULEHG   

7UDQVIHFWLRQ RI SODVPLG '1$ WR UHFLSLHQW FHOOV ZDV DFKLHYHG XVLQJ SRO\HWK\OHQLPLQH

3RO\VFLHQFHV  DQG WKH WUDQVIHFWHG FHOOV ZHUH LQFXEDWHG IRU  K DW  ƒ& IROORZHG E\

ELRFKHPLFDODQGELRORJLFDODVVD\VXQOHVVRWKHUZLVHVWDWHG

Immunohistochemistry (IHC))RUPDOLQIL[HGSDUDIILQHPEHGGHG ))3( WLVVXHVHFWLRQV

ȝP IURP6WRQ\%URRN8QLYHUVLW\5HVHDUFK+LVWRORJ\&RUH/DEDSSURYHGE\WKH,5%RI

6WRQ\ %URRN 8QLYHUVLW\ ZHUH H[DPLQHG E\ D PRGLILHG ,+& PHWKRG   $QWLJHQ UHWULHYDO

ZDV DFKLHYHG E\ ERLOLQJ WLVVXH VHFWLRQV IRU  PLQXWHV LQ 0 VRGLXP FLWUDWH S+  

6HFWLRQVZHUHEORFNHGIRURQHKRXULQ%6$DWURRPWHPSHUDWXUHDQGLQFXEDWHGLQUDEELW

DQWL003 DQWLERGLHV DW ƒ& RYHUQLJKW $IWHU ZDVKLQJ VDPSOHV ZHUH LQFXEDWHG ZLWK

+53FRQMXJDWHGDQWLUDEELW,J*DQGWKHQE\%LRWLQ;;7\UDPLGHDPSOLILFDWLRQ ,QYLWURJHQ 

DQG VWUHSWDYLGLQ+53 6WDLQHG VHFWLRQV ZHUH YLVXDOL]HG XVLQJ ¶GLDPLQHEHQ]LGLQH

WHWUDK\GURFKORULGH '$% DQGFRXQWHUVWDLQHGZLWKKHPDWR[\OLQ,+&VWDLQLQJZLWKRXWSULPDU\

DQWLERG\ZDVXVHGDVDQHJDWLYHFRQWURO

 Dot-Based Cell Migration Assay7UDQVIHFWHGFHOOVZHUHPL[HGZLWKDQHTXDOYROXPHRI

QHXWUDOL]HGW\SH,FROODJHQ PJPO RQLFH7KHFHOOFROODJHQPL[WXUH ȝORI[FHOOPO 

ZDV WKHQ GRWWHG RQWR D ZHOO SODWH $IWHU VROLGLILFDWLRQ RI FHOOFROODJHQ GRWV WKH

FHOOFROODJHQ KHPLVSKHUHV ZHUH FRYHUHG ZLWK ȝO RI FRPSOHWH PHGLD DQG LQFXEDWHG IRU 

KRXUV IROORZHG E\ VWDLQLQJ ZLWK QXFOHDU G\H +RHFKVW DQG FRXQWLQJ RI WKH PLJUDWHG FHOOV



Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.




XVLQJ1LNRQ1,6(OHPHQWVLPDJLQJVRIWZDUH 

Macrodissection and RNA extraction7XPRUFHOOVDWWKHLQYDVLYHIURQWDVZHOODVWXPRU

DGMDFHQWQRUPDOFHOOVZHUHPDFURGLVVHFWHGIURPIRXUFRQVHFXWLYHμP))3(KXPDQFRORQ

FDQFHU VHFWLRQV ZKLFK ZHUH FDUHIXOO\ DOLJQHG DQG PDUNHG ZLWK

LPPXQRKLVWRFKHPLVWU\VWDLQLQJ SHUIRUPHG E\ DQ H[SHULHQFHG SDWKRORJLVW  PL51$V ZHUH

WKHQH[WUDFWHGXVLQJPL51HDV\))3(.LW 4LDJHQ  

Statistical Analysis'DWDDUHH[SUHVVHGDVWKHPHDQ“VWDQGDUGHUURURIWULSOLFDWHV(DFK

H[SHULPHQWZDVUHSHDWHGDWOHDVWWLPHV  6WXGHQW¶VtWHVWDQGDQDO\VLVRIYDULDQWV $129$ 

ZHUHXVHGWRDVVHVVGLIIHUHQFHVZLWK 3  3 3

$OORWKHUPHWKRGVDUHDYDLODEOHDVVXSSOHPHQWDU\PDWHULDOVRQOLQH 
 




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.




5HVXOWV

MMP-14 is upregulated in various human cancers

 8SUHJXODWHG 003 H[SUHVVLRQ LQ FDQFHU FHOOV KDV EHHQ VKRZQ WR SURPRWH WXPRU

JURZWKDQGPHWDVWDVLV  $OWKRXJK003KDVEHHQUHSRUWHGWREHXSUHJXODWHGLQYDULRXV

KXPDQ FDQFHUV   WKHUH LV QR V\VWHPDWLF DQDO\VLV RI 003 H[SUHVVLRQ LQ GLIIHUHQW

FDQFHUV IURP GLIIHUHQW RUJDQV %\ PLQLQJ '1$ PLFURDUUD\ GDWDEDVHV DW 2QFRPLQH &DQFHU

3URILOLQJ 'DWDEDVH  H[SUHVVLRQ RI 003 LQ KXPDQ FDQFHU VSHFLPHQV ZDV DVVHVVHG 

:KHQ DVVLJQLQJ D S OLPLW D P\ULDG RI FDQFHUV GLVSOD\HG XSUHJXODWLRQ RI 003 DV

FRPSDUHGWRUHVSHFWLYHQRQPDOLJQDQWFRQWUROWLVVXHV  )LJ$   

 7RYDOLGDWH WKHGDWD PLQLQJUHVXOWVKXPDQEUHDVW FDQFHUVSHFLPHQVZHUH H[DPLQHGIRU

WKH H[SUHVVLRQ RI 003 E\ H[DPLQLQJ P51$ DQG SURWHLQ OHYHOV $ ODVHU FDSWXUH

PLFURGLVVHFWLRQ /&0 WHFKQLTXHZDVHPSOR\HGWRKDUYHVWEUHDVWFDUFLQRPDFHOOVDQGWXPRU

DGMDFHQW QRUPDO HSLWKHOLDO FHOOV DV ZH SUHYLRXVO\ HPSOR\HG   IROORZHG E\ UHDOWLPH

573&5IRUWKHP51$RI0032XUGDWDVKRZVWKDW003LVVHOHFWLYHO\H[SUHVVHGLQ

KXPDQ EUHDVW GXFWDO FDUFLQRPD LQ VLWX '&,6  DQG LQ LQYDVLYH GXFWDO FDUFLQRPD EXW QRW LQ

QRUPDO HSLWKHOLDO FHOOV ([SUHVVLRQ RI 003 LQ LQYDVLYH GXFWDO FDUFLQRPD LV HOHYDWHG DV

FRPSDUHG WR '&,6 DOWKRXJK WKHUH LV QR VWDWLVWLFDO VLJQLILFDQFH DPRQJ WKHP )LJ%  7R

FRQILUP WKLV REVHUYDWLRQ LPPXQRKLVWRFKHPLVWU\ ,+&  ZDV SHUIRUPHG LQ WKH FRUUHVSRQGLQJ

KXPDQ EUHDVW FDQFHU VSHFLPHQV XVLQJ DQ DQWL003 DQWLERG\ ,Q DJUHHPHQW ZLWK WKH

UHDOWLPH573&5GDWD003SURWHLQZDVGHWHFWHGRQO\LQKXPDQEUHDVWFDQFHUFHOOVEXW

QRWLQDGMDFHQWQRUPDOHSLWKHOLDOFHOOV )LJ& 

 7RIXUWKHUFRQILUPRXUGDWDPLQLQJUHVXOWVVKRZLQJWKDWWKHXSUHJXODWLRQRI003LVD




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.




JHQHUDOSKHQRPHQRQLQFDQFHUKXPDQFRORQFDQFHUVSHFLPHQVZHUHH[DPLQHGE\,+&XVLQJ

WKH DQWL003 DQWLERG\ :H REVHUYHG WKDW 003 LV PLQLPDOO\ H[SUHVVHG LQ QRUPDO

FRORQLF PXFRVDO FHOOV EXW LQFUHDVHG LQWHQVLW\ RI VWDLQLQJ LV IRXQG LQ FDQFHU FHOOV DW WKH

LQYDVLYH IURQW )LJ'(  7KXV RXU VWXG\ LQGLFDWHV WKDW 003 LV XSUHJXODWHG LQ KXPDQ

FDQFHUVDQGWKHH[SUHVVLRQOHYHORI003LVFRUUHODWHGZLWKWKHLQYDVLYHVWDWXVRIKXPDQ

FDQFHU 

Computational prediction of miRNA-181s as potential regulators of MMP-14

003 LV XSUHJXODWHG LQ KXPDQ FDQFHUV KRZHYHU WKH UHJXODWRU\PHFKDQLVP RI

003 UHPDLQV WR EH IXOO\ FKDUDFWHUL]HG :KHQ 003*)3 FKLPHULF

F'1$ 003*)3  FRQWDLQLQJ WKH 


875 RI 003 003*)3
875  ZDV

WUDQVIHFWHG LQWR PLQLPDOO\ LQYDVLYH KXPDQ EUHDVW FDQFHU 0&) DQG KLJKO\ LQYDVLYH

0'$0% FHOOV ZH QRWLFHG WKDW WKH H[SUHVVLRQ RI 003*)3 LQ 0&) FHOOV LV

UHODWLYHO\ ZHDN DV FRPSDUHG WR WKDW LQ 0'$0% FHOOV )LJ $ WRS SDQHO  7KLV

GLIIHUHQFH KRZHYHU ZDV QRW VHHQ LQ WKHVH FHOOV ZKHQ WUDQVIHFWHG ZLWK WKH 003*)3

FKLPHUD WKDW GRHV QRW FRQWDLQ WKH 


875 )LJ$ERWWRP SDQHO  7KLV REVHUYDWLRQ LV

UHSURGXFHGLQSURVWDWHFDQFHUFHOOOLQHVRI/1&D3 PLQLPDOO\LQYDVLYH DQG'8DQG3&

KLJKO\ LQYDVLYH )LJ%  VXJJHVWLQJ WKDW WKH ¶875 RI 003 P51$ PD\ FRQWDLQ D

UHJXODWRU\ VHTXHQFH V  WKDW LV UHVSRQVLEOH IRU GLIIHUHQWLDO H[SUHVVLRQ RI 003 LQ FDQFHU

OLQHV 

,WLVNQRZQWKDWWKH
875RIWHQFRQWDLQVPL51$UHVSRQVHHOHPHQWV 05(V ZKLFKDUH

VHTXHQFHVWRZKLFKPL51$VELQG7RLGHQWLI\SXWDWLYH05(VZLWKLQWKH¶875RI003

P51$ ZH XVHG 7DUJHW6FDQ WR VHDUFK IRU SRWHQWLDO 05(V ZLWKLQ WKH 
875 7KLV DQDO\VLV




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.




UHYHDOHG PXOWLSOH 05(V WKDW SRWHQWLDOO\ LQWHUDFW ZLWK PL51$     DQG

V 7R LQFUHDVH WKH SUREDELOLW\ RI SRVWXODWHG 05(V ZLWKLQ WKH 
875 DQRWKHU PL51$

SUHGLFWLRQ DOJRULWKP PL5DQGD ZDV HPSOR\HG  %\ DQDO\]LQJ SUHGLFWHG 05(V IURP ERWK

7DUJHW6FDQ DQG PL5DQGD RQO\ PL5 DQG V ZHUH IRXQG WR RYHUODS EHWZHHQ WKH WZR

FRPSXWDWLRQDODQDO\VHV7KH05(RIPL5ZLWKLQWKH
875EHWZHHQQWDQGQW

UHFHLYHGWKHKLJKHVWSUHGLFWLRQVFRUHIRUELQGLQJWRPL5V )LJ& DQGKHQFHPL5V

ZHUHIXUWKHUFKDUDFWHUL]HG   

Interference of ectopically expressed MMP-14 by miR-181a-5p 

6LQFHPL5LVRIRUPVFRQWDLQLGHQWLFDOVHHGVHTXHQFHWRWKH003
875 )LJ& 

PL5D DORQJ ZLWK WKH  ES IODQNLQJ UHJLRQV DW ERWK VLGHV RI PL5D ZDV

DPSOLILHG IURP WKH FKURPRVRPDO '1$ DQG WKH UHVXOWDQW '1$ ZDV FORQHG LQWR DQ

0'+3*.*)3 UHWURYLUDO YHFWRU WKDW FRQWDLQV D *)3 UHSRUWHU $GGJHQH  %DVHG RQ

UHFHQWPL51$QRPHQFODWXUHJXLGHOLQHV KWWSZZZPLUEDVHRUJ ZHGHVLJQDWHGWKHPDWXUH

VHTXHQFH RI KVDPL5D LQ WKH YHFWRU DV PL5DS*)3 8VLQJ D VLPLODU VWUDWHJ\

PL5 WKDW ZDV QRW SUHGLFWHG WR ELQG WKH 003 


875 ZDV DOVR FORQHG LQ

0'+3*.*)3 UHWURYLUDO YHFWRU DV D FRQWURO PL5*)3  (FWRSLF H[SUHVVLRQ RI

PL5DS ZDV H[DPLQHG E\ WUDQVLHQW WUDQVIHFWLRQ RI WKH F'1$V HQFRGLQJ

PL5DS*)3 DV ZHOO DV FRQWURO YHFWRUV LQWR &26 FHOOV ZKLFK GR QRW H[SUHVV

GHWHFWDEOH HQGRJHQRXV 003   0DWXUH PL5DS DQG  ZHUH GHWHFWHG LQ WKH

WUDQVIHFWHG &26 FHOOV E\ D UHDOWLPH 573&5 DSSURDFK ZLWK  DQG IROG LQFUHDVHV RI

PL5DS DQG  UHVSHFWLYHO\ DV FRPSDUHG WR YHFWRU FRQWURO )LJ$  LQGLFDWLQJ

PDWXUHPL5DSDQGDUHHIILFLHQWO\SURGXFHGLQWKHWUDQVIHFWHGFHOOV  




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.




:H WKHQ H[DPLQHG ZKHWKHU PL5DS FDQ DIIHFW HFWRSLFDOO\ H[SUHVVHG 003 LQ

&26 FHOOV E\ FRWUDQVIHFWLQJ 003


875 ZLWK PL5FRQWURO PL5DS*)3 DQG

PL5*)3 UHVSHFWLYHO\ 7R LQFUHDVH WKH H[SUHVVLRQ HIILFLHQF\ IRU ERWK PL51$ DQG

003
875 &26 FHOOV ZHUH ILUVW PDGH WR VWDEO\ H[SUHVV HDFK PL51$ DQG ZDV

IROORZHG E\ WUDQVLHQW WUDQVIHFWLRQ RI 003


875 F'1$ DQG YHFWRU FRQWURO  (FWRSLF

H[SUHVVLRQ RI PL5DS EXW QRW PL5FRQWURO DQG PL5 OHG WR UHGXFHG 003

P51$ DQG SURWHLQ OHYHOV )LJ%&  VXJJHVWLQJ WKDW 003 P51$LV GRZQUHJXODWHG E\

PL5DS 

7RIXUWKHUGHWHUPLQHZKHWKHUWKHORVVRI003H[SUHVVLRQLQERWKP51$DQGSURWHLQ

OHYHOV E\ PL5DS LV GXH WR WKH H[LVWHQFH RI WKH 003 
875 ZH HPSOR\HG D

SUHYLRXVO\JHQHUDWHGSODVPLG'1$HQFRGLQJRQO\WKHRSHQUHDGLQJIUDPHRI003DQG

WKHUHIRUH ODFNV WKH 


875 RI 003   :HVWHUQ EORWWLQJ UHYHDOHG WKDW WKHUH ZDV QR

GLVWLQFWGLIIHUHQFHLQHFWRSLF003H[SUHVVLRQEHWZHHQWKHFHOOVWKDWRYHUH[SUHVVHGHLWKHU

PL5DS RU PL5FRQWURO )LJ'  FRQILUPLQJ WKH H[LVWHQFH RI D PL5 UHVSRQVH

HOHPHQWZLWKLQWKH
875 

7R IXUWKHU YDOLGDWH WKH UROH RI PL5DS LQ UHJXODWLRQ RI 003 H[SUHVVLRQ ZH

JHQHUDWHG 8 SURPRWHUGULYHQ VSRQJH WR GRZQUHJXODWH PL5DS )LJ(  :KHQ WKH

VSRQJHVZHUHVWDEO\LQWURGXFHGLQWR0&)FHOOVZKLFKH[SUHVVDKLJKOHYHORIHQGRJHQRXV

PL5DS ZH IRXQG WKDW WKH VSRQJHV HIILFLHQWO\ UHGXFHG WKH PL5DS OHYHO LQ WKH

FHOOVDVFRPSDUHGWRWKHFRQWURO )LJ)  

7R GHWHUPLQH LI UHGXFHG HQGRJHQRXV PL5DS E\ WKH VSRQJHV OHDGV WR HQKDQFHG

003H[SUHVVLRQHQGRJHQRXVDQGHFWRSLFDOO\H[SUHVVHG003ZHUHH[DPLQHG,WKDV




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.




EHHQ UHSRUWHG WKDW K\SR[LD LQGXFHV 003 H[SUHVVLRQ   :KHQ 0&) FHOOV VWDEO\

H[SUHVVLQJ WKH VSRQJHV ZHUH FXOWXUHG XQGHU K\SR[LD  2  IRU  KRXUV HQGRJHQRXV

003LQFUHDVHGLQFHOOVH[SUHVVLQJPL5DSVSRQJHVDVFRPSDUHGWRFRQWUROVSRQJHV

)LJ* 7RIXUWKHUYDOLGDWHWKHUROHRIWKHPL5DSLQUHJXODWLQJ003H[SUHVVLRQ

WKHHIIHFWRIPL5DSRQHFWRSLF003ZDVH[DPLQHG0&)FHOOVVWDEO\H[SUHVVLQJ

WKH VSRQJHV ZHUH WUDQVIHFWHG ZLWK 003*)3


875 DQG VXEVHTXHQWO\ DQDO\]HG IRU WKH

003*)3P51$DQGSURWHLQH[SUHVVLRQV(FWRSLFH[SUHVVLRQRIPL51$DSVSRQJH

EXWQRWWKHPL5FRQWUROVSRQJHOHGWRXSUHJXODWHG003*)3LQERWKP51$DQGSURWHLQ

OHYHOV )LJ+,  IXUWKHU VXJJHVWLQJ WKDW 003 LV QHJDWLYHO\ UHJXODWHG E\

PL51$DS

Direct interaction of miR-181a-5p in the MMP-14 3'UTR

7R GHWHUPLQH LI PL5DS WDUJHWV WKH 003 


875 GLUHFWO\ ZH XWLOL]HG D

OXFLIHUDVH UHSRUWHU JHQH IXVHG WR D VHTXHQFH RI WKH 


875 RI 003 WKDW FRQWDLQV WKH

SUHGLFWHG PL5DS UHVSRQVH HOHPHQW /XF


875  $V D FRQWURO WKH SUHGLFWHG

PL5DS UHVSRQVH HOHPHQW  QW  ZDV FRQYHUWHG WR WKH FRPSOHPHQWDU\ VHTXHQFH WR

HOLPLQDWH SRWHQWLDO PL5DS ELQGLQJ E\ XVLQJ D VLWHGLUHFW PXWDJHQHVLV DSSURDFK

/XF
875PX    ([SUHVVLRQ RI PL5DS VLJQLILFDQWO\ UHGXFHG OXFLIHUDVH DFWLYLW\ RI

/XF
875 ZKHUHDV LW KDG QR HIIHFW RQ FHOOV H[SUHVVLQJ PL5FRQWURO RU /XF
875PX

)LJ$ :KHQ/XF
875UHSRUWHUJHQHZDVWUDQVIHFWHGLQWR0&)FHOOVVWDEO\H[SUHVVLQJ

PL5DS VSRQJHV  WKH OXFLIHUDVH DFWLYLW\ VLJQLILFDQWO\ LQFUHDVHG DV FRPSDUHG WR

PL5FRQWURO VSRQJH LQIHFWHG FHOOV ZKHUHDV OXFLIHUDVH DFWLYLW\ IURP /XF


875PX ZDV QRW

DIIHFWHGE\PL5DSVSRQJH )LJ% +HQFHWKHREVHUYHGGRZQUHJXODWLRQRI003




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.




E\ PL5DS GHSHQGV GLUHFWO\ RQ D VLQJOH FRJQDWH UHFRJQLWLRQ VLWH LQ WKH 
875 RI

003P51$ 

8VLQJ DQ LPDJLQJEDVHG DVVD\ ZH REVHUYHG WKDW IOXRUHVFHQFH LQWHQVLW\ LQ &26 FHOOV

FRH[SUHVVLQJ003*)3
875ZLWKPL5DS ODFNLQJ*)3 VLJQLILFDQWO\GHFUHDVHG

DV FRPSDUHG WR FRQWURO )LJ&'  &RQYHUVHO\ IOXRUHVFHQFH LQWHQVLW\ RI

003*)3
875LQ0&)FHOOVVWDEO\H[SUHVVLQJWKHPL5DSVSRQJHVLJQLILFDQWO\

LQFUHDVHGDVFRPSDUHGWRWKHFRQWUROVSRQJH )LJ( 

Effect of functional MMP-14 by miR-181a-5p

:H SUHYLRXVO\ UHSRUWHG WKDW IXQFWLRQDO 003 HQKDQFHV SURWHRO\WLF DFWLYLW\ DQG FHOO

PLJUDWRU\DELOLW\  003DVHFUHWRU\003WKDWSURPRWHVFDQFHULQYDVLRQDQGPHWDVWDVLV

KDV EHHQ XVHG DV DQ LQGLFDWRU IRU IXQFWLRQDO 003    :H WKHQ H[DPLQHG ZKHWKHU

UHGXFHG003H[SUHVVLRQE\PL5DSUHVXOWVLQGHFUHDVHG003DFWLYDWLRQDQGFHOO

PLJUDWLRQ&RH[SUHVVLRQRI003
875ZLWKPL5DS*)3LQ&26FHOOVUHVXOWHG

LQUHGXFHGSUR003DFWLYDWLRQDVGHPRQVWUDWHGE\GHFUHDVHGLQWHUPHGLDWH LQW003 DQG

IXOO\ DFWLYDWHG DFW003  003 DORQJ ZLWK LQFUHDVHG ODWHQW 003 SUR003  DV

FRPSDUHG WR FHOOV H[SUHVVLQJ PL5FRQWURO DQG PL5 )LJ $  7KLV GHFUHDVH FRUUHODWHV

ZLWK UHGXFHG 003 H[SUHVVLRQ )LJ&  6LQFH 7,03 LV D QDWXUDO LQKLELWRU RI

003   ZH H[DPLQHG LI WKH GHFUHDVHG DFWLYLW\ RI 003 E\ PL5DS LV GXH WR

XSUHJXODWHG 7,03 2XU :HVWHUQ EORWWLQJ GDWD UXOHV RXW WKDW SRVVLELOLW\ EHFDXVH

PL5DSGRHVQRWHQKDQFH7,03H[SUHVVLRQLQ0'$0%FHOOV )LJ%  

&RQVLVWHQWZLWK003DFWLYDWLRQHFWRSLFH[SUHVVLRQRIPL5DS*)3DORQJZLWK

003
875 LQ &26 FHOOV OHG WR D GHFUHDVH LQ FHOO PLJUDWLRQ )LJ&'  ,PSRUWDQWO\




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.




PL51$ZDVXQDEOHWRLQKLELWPLJUDWLRQLQWKH&26FHOOVH[SUHVVLQJ003
875

)LJ&'  VXJJHVWLQJ WKDW PL5DS LV D NH\ IDFWRU LQ FRQWURO RI 003 H[SUHVVLRQ

ZKRVHGRZQUHJXODWLRQUHVXOWVLQGHFUHDVHGSURWHRO\WLFSRWHQWLDODQGFHOOPLJUDWRU\DELOLW\  

Inverse correlation of miR-181a-5p and MMP-14 in human cancer cell lines and human

cancer specimens

,W KDV EHHQ UHSRUWHG WKDW XSUHJXODWHG 003 LV RIWHQ REVHUYHG LQ LQYDVLYH KXPDQ

FDQFHUV DQG FRUUHODWHV ZLWK DJJUHVVLYHQHVV RI KXPDQ FDQFHU FHOO OLQHV   7R

LQYHVWLJDWH WKH FRUUHODWLRQ EHWZHHQ WKH HQGRJHQRXV PL5DS OHYHO DQG 003

H[SUHVVLRQLQKXPDQFDQFHUFHOOOLQHVZHVXUYH\HGKXPDQFDQFHUFHOOOLQHVXVLQJUHDOWLPH

573&5DQG:HVWHUQEORWWLQJ  $VH[SHFWHG003H[SUHVVHVDWDUHODWLYHO\KLJKOHYHOLQ

DJJUHVVLYH FDQFHU OLQHV LQFOXGLQJ +7 0'$0% DQG EUHDVW FDQFHU VWHP 6.UG

FHOOV )LJ$% ,QWHUHVWLQJO\PL5DSH[SUHVVLRQLVVLJQLILFDQWO\ORZHULQWKHVHFHOOVDV

FRPSDUHG WR OHVV DJJUHVVLYH FDQFHU FHOO OLQHV VXFK DV 0&) DQG 6.%5 OLQHV )LJ&  

7R GHWHUPLQH FOLQLFDO VLJQLILFDQFH ZH H[DPLQHG WKH FRUUHODWLRQ EHWZHHQ PL5DS DQG

003 LQ KXPDQ FDQFHU VSHFLPHQV  6LQFH 003 LV PLQLPDOO\ H[SUHVVHG LQ QRUPDO

SULPDU\FRORQLFPXFRVDOFHOOVEXWDQLQFUHDVHGLQWHQVLW\RIVWDLQLQJLVIRXQGLQFDQFHUFHOOV

ORFDWHGDWWKHLQYDVLYHIURQW )LJ( ZHKDUYHVWHGWXPRUFHOOVDWWKHLQYDVLYHIURQWRIKXPDQ

FRORQ FDQFHU VSHFLPHQV DV ZHOO DV WXPRU DGMDFHQW QRUPDO HSLWKHOLDO FHOOV XVLQJ D

PDFURGLVVHFWLRQWHFKQLTXHIROORZHGE\UHDOWLPH573&5IRUPL5DS&RQVLVWHQWZLWK

WKHREVHUYDWLRQLQFHOOOLQHV )LJ& PL5DSLVGRZQUHJXODWHGLQLQYDVLYHWXPRUFHOOV

DV FRPSDUHG WR WXPRU DGMDFHQW QRUPDO HSLWKHOLDO FHOOV )LJ'  7KHVH GDWD VXJJHVW WKDW

H[SUHVVLRQ RI 003 LQ KXPDQ LQYDVLYH FDQFHU LV DIIHFWHG E\ PL5DS OHYHOV 7R




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.




IXUWKHU GHOLQHDWH WKH FDXVDO UHODWLRQVKLS EHWZHHQ PL5DS DQG 003 H[SUHVVLRQ LQ

FDQFHUSURJUHVVLRQZHXWLOL]HG0'$0%FHOOVEHFDXVHWKLVFHOOOLQHLVKLJKO\LQYDVLYH

DQG H[SUHVVHV D KLJK OHYHO RI 003 DV FRPSDUHG WR RWKHU FDQFHU FHOO OLQHV :KHQ

0'$0% FHOOV ZHUH WUDQVLHQWO\ WUDQVIHFWHG ZLWK PL5DS*)3 RU

PL5FRQWURO )LJ(  WKH HQGRJHQRXV 003 H[SUHVVLRQ DW ERWK P51$ DQG SURWHLQ OHYHO

ZDVVLJQLILFDQWO\UHGXFHG )LJ) 7KLVFRUUHODWLRQZDVDOVRUHSURGXFHGLQ+7FHOOVWKDW

H[SUHVVDKLJKOHYHORIHQGRJHQRXV003 )LJ*+ 

6SHFLILFGRZQUHJXODWLRQRI003H[SUHVVLRQKDVEHHQVKRZQWRVLJQLILFDQWO\LQKLELW

FDQFHUFHOOPLJUDWLRQDQGLQYDVLRQHYHQWKRXJKRWKHU003VFRQWLQXHGWREHH[SUHVVHG  

7KHUHIRUH ZH DVNHG ZKHWKHU PL5DS FDQ DIIHFW FDQFHU FHOO PLJUDWLRQ ([SUHVVLRQ RI

PL5DS LQ 0'$0% FHOOV UHVXOWHG LQ D VLJQLILFDQW GHFUHDVH LQ FHOO PLJUDWLRQ DV

H[DPLQHG E\ D 7UDQVZHOO FKDPEHU PLJUDWLRQ DVVD\ )LJ$  7KH DELOLW\ RI PL5DS WR

UHGXFH FHOO PLJUDWLRQ ZDV IXUWKHU FRQILUPHG LQ +7 FHOOV HFWRSLFDOO\ H[SUHVVLQJ

PL5DS )LJ%  

6LQFH FHOO PLJUDWLRQ LV D FULWLFDO GHWHUPLQDWLRQ RI FDQFHU LQYDVLRQ D WKUHHGLPHQVLRQDO

'  LQYDVLRQ DVVD\   ZDV XWLOL]HG WR GHWHUPLQH LI RYHUH[SUHVVLRQ RI PL5DS LQ

0'$0% FHOOV DQG +7 FHOOV FRXOG LQKLELW FHOO LQYDVLRQ $V H[SHFWHG ZKHQ

RYHUH[SUHVVLQJ PL5DS FHOO LQYDVLYH DELOLW\ ZDV GUDPDWLFDOO\ GHFUHDVHG LQ ERWK FHOO

OLQHV )LJ&'  

%HFDXVH 003 LV D WUDQVPHPEUDQH SURWHDVH ZH QH[W DVNHG LI UHGXFHG FDQFHU FHOO

PLJUDWLRQDQGLQYDVLRQE\PL5DSZHUHGXHWRWKHORVVRIFHOOVXUIDFH003%\FHOO

VXUIDFH ELRWLQ\ODWLRQ DVVD\ ZH IRXQG WKDW FHOO VXUIDFH 003 ZDV PDUNHGO\ UHGXFHG LQ




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.




+7FHOOVHFWRSLFDOO\H[SUHVVLQJKLJKOHYHOVRIPL5DS )LJ( 

Overexpression of miR-181a-5p attenuates in vivo invasion and angiogenesis

003LVOLQNHGWRHQKDQFHGFDQFHULQYDVLRQ  7RGLUHFWO\H[DPLQHLIPL5DS

LV FDSDEOH RI LQKLELWLRQ RI 003PHGLDWHG FDQFHU FHOO LQYDVLRQ WKURXJK EDVHPHQW

PHPEUDQH in vivo WKH FKLFN FKRULRDOODQWRLF PHPEUDQH &$0  LQYDVLRQ DVVD\ ZDV

HPSOR\HG   7KH &$0 FRQVLVWV RI WKH FKRULRQLF HSLWKHOLXP DQG XQGHUO\LQJ DOODQWRLF

PHPEUDQH WKDW LV SULPDULO\ PDGH RI W\SH ,9 FROODJHQ   ZKLFK VLPXODWHV WKH EDVHPHQW

PHPEUDQH RI KXPDQ HSLWKHOLXP    ,QYDVLRQ RI FDQFHU FHOOV WKURXJK WKH HSLWKHOLXP DQG

EDVHPHQWPHPEUDQHRIWKHXSSHU&$0LQWRFRQQHFWLYHWLVVXHZDVH[DPLQHGE\+HPDWR[\OLQ

DQG(RVLQ>+ (@VWDLQLQJ  PL5FRQWURO0'$0%FHOOVWKDWZHUHORDGHGRQWRWKH&$0

LQYDGHG LQWR WKH FRQQHFWLYH WLVVXHV WKURXJK WKH EUHDFKHG EDVHPHQW PHPEUDQH )LJ)  ,Q

FRQWUDVW 0'$0% FHOOV VWDEO\ H[SUHVVLQJ PL5DS IDLOHG WR FURVV WKURXJK WKH

EDVHPHQWPHPEUDQH,QDGGLWLRQPRUHQHZEORRGYHVVHOVXQGHUQHDWKWKH&$0FDQEHIRXQG

LQ PL5FRQWURO FHOOV DV FRPSDUHG WR PL5DS FHOOV 7KLV UHGXFWLRQ LQ LQYDVLRQ DORQJ

ZLWKWKHGHFUHDVHLQDQJLRJHQHVLVVXJJHVWVWKDWPL5DSLVHIIHFWLYHDWLQWHUIHULQJZLWK

WKH in vivo LQYDVLYH DELOLW\ RI 0'$0% FHOOV ZKLFK QRUPDOO\ H[SUHVV KLJK OHYHOV RI

003      

7R IXUWKHU FKDUDFWHUL]H WKH DQWLDQJLRJHQLF DFWLYLW\ RI PL5DS WKURXJK

GRZQUHJXODWLRQ RI 003 H[SUHVVLRQ +7 FHOOV ZHUH HPSOR\HG 6LQFH PLQLPDO

PL5DSLVSUHVHQWLQ+7FHOOVZHVWDEO\H[SUHVVHGPL5DSLQ+7FHOOV

DQG DSSOLHG WKH FHOOV RYHU FKRULRDOODQWRLF PHPEUDQHV YLD VSRQJHV DV SUHYLRXVO\ GHVFULEHG

   PL5DS EXW QRW PL5FRQWURO VWDWLVWLFDOO\ LPSDLUHG QHZ EORRG YHVVHO




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.




IRUPDWLRQLQGXFHGE\+7FHOOV )LJ*  

7DNHQ WRJHWKHU ZH IRU WKH ILUVW WLPH GHPRQVWUDWH WKDW PL5DS LV D FULWLFDO

UHJXODWRU IRU 003 H[SUHVVLRQ DQG FDQ DIIHFW 003PHGLDWHG FDQFHU FHOO PLJUDWLRQ

LQYDVLRQDQGDQJLRJHQHVLV  
 




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.




'LVFXVVLRQ

,QWKLVVWXG\ZHILUVWYDOLGDWHGWKDW003LVKLJKO\XSUHJXODWHGLQKXPDQEUHDVWDQG

FRORQFDQFHUV:HWKHQGHPRQVWUDWHGWKDWPL5DSLVLQYHUVHO\FRUUHODWHGZLWK003

H[SUHVVLRQDQGWKHLQYDVLYHFDSDFLW\RIFDQFHUFHOOOLQHV:HDOVRLGHQWLILHGWKHPL5DS

WDUJHW VHTXHQFH ZLWKLQ WKH 003 


875 WKDW LV UHVSRQVLEOH IRU WKH VWDELOLW\ RI 003

P51$(FWRSLFH[SUHVVLRQRIPL5DSUHVXOWHGLQGRZQUHJXODWLRQRIERWKHQGRJHQRXV

DQG H[RJHQRXV 003 H[SUHVVLRQ OHDGLQJ WR GHFUHDVHG FHOO PLJUDWLRQ LQYDVLRQ DQG

DQJLRJHQHVLV $OWKRXJK 003 LV WUDQVFULSWLRQDOO\ UHJXODWHG E\ DFWLYDWLQJ WKH JHQH¶V

SURPRWHU   WKH HIIHFW RI WKH VWDELOLW\ RI 003 P51$ LV DQRWKHU NH\ UHJXODWRU\

PHFKDQLVP LQ FRQWUROOLQJ 003 H[SUHVVLRQ +HQFH RXU REVHUYDWLRQV XQUDYHO WKH

SRVWWUDQVFULSWLRQDOUHJXODWRU\PHFKDQLVPIRU003H[SUHVVLRQ 

PL51$VFDQLQGXFHJHQHH[SUHVVLRQE\ELQGLQJWRWKH¶875RIWKHSURPRWHUUHJLRQRI

WDUJHWHG JHQHV RU UHGXFH JHQH H[SUHVVLRQ E\ ELQGLQJ WR WKH ¶875 RI WKH WDUJHW JHQH DQG

DOORZLQJ IRU P51$ GHJUDGDWLRQ RU SUHYHQWLQJ P51$ IURP EHLQJ WUDQVODWHG 2XU GDWD

LQGLFDWHV WKDW PL5DS QHJDWLYHO\ DIIHFWV 003 H[SUHVVLRQ WKURXJK ELQGLQJ WR WKH

¶875RI003%HFDXVH003P51$LVUHGXFHGE\PL5DSLWLVDVVXPHGWKDW

PL5DS LQGXFHV 003 P51$ GHJUDGDWLRQ UDWKHU WKDQ EORFNLQJ 003 SURWHLQ

WUDQVODWLRQ  6LQFHXSUHJXODWLRQRI003GLUHFWO\DVVRFLDWHVZLWKFDQFHUDJJUHVVLYHQHVV

LQGXFWLRQ RI HQGRJHQRXV PL5DS SURYLGHV D SRWHQWLDO DSSURDFK WR SUHYHQW FDQFHU

LQYDVLRQ DQG PHWDVWDVLV  +RZHYHU LW VKRXOG EH SRLQWHG RXW WKDW WKH UROH RI PL5V LQ

FDQFHULVVWLOOFRQWURYHUVLDOGHSHQGLQJRQWKHWXPRUW\SH 

6HYHUDO VWXGLHV KDYH LQGLFDWHG WKDW PL5V VHUYH DV WXPRUSURPRWLQJ JHQHV EXW WKH




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.




IXQFWLRQRIPL5DSLVWXPRUW\SHVSHFLILFPL5DSZDVIRXQGWREHXSUHJXODWHGLQ

JDVWULF DQG OLYHU FDQFHUV   DQG LQ WZR VHSDUDWH VWXGLHV PL5DS RYHUH[SUHVVLRQ

ZDV UHSRUWHG WR HQKDQFH RYDULDQ OLYHU DQG EUHDVW FDQFHU SURJUHVVLRQ WKURXJK GLIIHUHQW

PHFKDQLVPV   +RZHYHU PL51$V KDYH DOVR EHHQ VXJJHVWHG DV WXPRU

VXSSUHVVRUV LQ VHYHUDO W\SHV RI KXPDQ FDQFHUV LQFOXGLQJ OHXNHPLD JOLRPD DQG RUDO

VTXDPRXVFHOOFDUFLQRPD  7KHVHFRQWURYHUVLDOREVHUYDWLRQVVXJJHVWWKHFRPSOH[LW\RI

PL51$VDQGWKHIXQFWLRQRIVSHFLILFPL51$VFDQGLIIHUPDUNHGO\GHSHQGLQJRQWXPRUW\SHV

,W LV ZRUWK QRWLQJ WKDW PRVW RI WKHVH VWXGLHV XVH WKH EXON RI KXPDQ WXPRU WLVVXHV ZKLFK

FRQWDLQ QRW RQO\ WXPRU FHOOV EXW DOVR YDULRXV VWURPDO FHOO W\SHV DQG DGMDFHQW QRUPDO

HSLWKHOLDOFHOOV7KLVPD\QRWIDLWKIXOO\UHSUHVHQWWKHH[SUHVVLRQOHYHORIVSHFLILFPL51$VLQ

FDQFHU,QGHHGRXUGDWDVXSSRUWWKLVQRWLRQE\VKRZLQJWKDWPL5DSDQG003DUH

GLIIHUHQWLDOO\H[SUHVVHGLQLQYDVLYHWXPRUFHOOVDQGWXPRUDGMDFHQWQRUPDOHSLWKHOLDOFHOOV 

$OWKRXJKWKLVVWXG\IRFXVHVRQPL5DSEHFDXVHLWUHFHLYHGWKHKLJKHVWSUREDELOLW\

VFRUH E\ FRPSXWDWLRQDO DQDO\VLV RWKHU PL51$V PD\ DOVR DIIHFW 003 H[SUHVVLRQ

$NDQXPD HW DO UHSRUWHG WKDW PL5 UHGXFHV 003 H[SUHVVLRQ OHDGLQJ WR GHFUHDVHG

FDQFHUFHOOPLJUDWLRQDQGLQYDVLRQ  6LQFHZHDOVRLGHQWLILHGDPL5UHVSRQVHHOHPHQW

ZLWKLQ WKH 
875 RI 003 P51$ E\ PL51$ SUHGLFWLRQ DOJRULWKPV $NDQXPD
V UHSRUW

VXSSRUWV RXU ELRLQIRUPDWLFV DQDO\VLV $QRWKHU PL51$ PL5 ZDV UHFHQWO\ UHSRUWHG WR

GRZQUHJXODWH 003 H[SUHVVLRQ E\ WDUJHWLQJ WKH 


875 UHVXOWLQJ LQ GHFUHDVHG FHOOXODU

LQYDVLRQDQGPHWDVWDVLVLQQHXUREODVWRPDFHOOV  $OWKRXJKWZRVHSDUDWHPL51$SUHGLFWLRQ

DOJRULWKPVZHUHHPSOR\HGWRLGHQWLI\PL51$UHVSRQVHHOHPHQWVZLWKLQWKH003
875

LQ RXU VWXG\ ZH GLG QRW REVHUYH WKH PL5 UHVSRQVH HOHPHQW ZLWKLQ WKH 003 
875 




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.




,QWHUHVWLQJO\ PL5 ZDV DOVR UHSRUWHG WR GLUHFWO\ WDUJHW &'+ WKH (FDGKHULQHQFRGLQJ

P51$ OHDGLQJ WR LQFUHDVHG FHOO PRWLOLW\ DQG LQYDVLYHQHVV ZKLFK XOWLPDWHO\ FRQWULEXWHV WR

PHWDVWDVLV    6LQFHPRXQWLQJHYLGHQFHLQGLFDWHVWKDW003FOHDYHV(FDGKHULQDWWKH

FHOOVXUIDFHOHDGLQJWRHQKDQFHGFDQFHUDJJUHVVLYHQHVV  ERWKUHSRUWVVHHPVFRQWUDGLFWRU\

XQGHUSDWKRORJLFDOFRQGLWLRQV4XHVWLRQVUHPDLQDVWRZKHWKHUWKHVHPL51$VV\QHUJLVWLFDOO\

RULQGLYLGXDOO\DIIHFW003JHQHH[SUHVVLRQOHDGLQJWRUHGXFHGFHOOLQYDVLRQ 

8SUHJXODWLRQ RI 003 LQ FDQFHU KDV EHHQ UHFRJQL]HG DV RQH RI WKH FULWLFDO

PHFKDQLVPVRIFDQFHUGLVVHPLQDWLRQ7KHH[SUHVVLRQRI003KDVEHHQGHWHFWHGLQWXPRU

FHOOVDQGDGMDFHQWVWURPDOFHOOVLQDYDULHW\RIKXPDQWXPRUV  8VLQJin situK\EULGL]DWLRQ

WHFKQLTXHV VHYHUDO UHSRUWV KDYH GHPRQVWUDWHG WKDW 003 LV PRVWO\ H[SUHVVHG LQ VWURPDO

FHOOV ZLWKLQ WXPRUV RU H[SUHVVHG LQ ERWK FDQFHU FHOOV DQG VXUURXQGLQJ ILEUREODVWV DQG

PDFURSKDJHV  ,QRXU,+&ZHIRXQG003H[FOXVLYHO\H[SUHVVHGLQFDQFHUFHOOV

2XU UHDOWLPH 573&5 GDWD H[DPLQLQJ ODVHU PLFURGLVVHFWHG FDQFHU FHOOV VXSSRUWV RXU ,+&

GDWD,QWHUHVWLQJO\ZHIRXQGWKDW003H[SUHVVHVDWDORZOHYHOLQSULPDU\FRORQFDQFHU

FHOOVZKHUHDVH[SUHVVLRQRI003GUDPDWLFDOO\LQFUHDVHVLQFRORQFDQFHUFHOOVORFDWHGDW

WKH LQYDVLYH IURQW RU WKRVH WKDW KDYH DOUHDG\ LQYDGHG LQWR WKH VXEPXFRVD 6LQFH 003

SOD\VDFULWLFDOUROHLQFDQFHUFHOO(07  RXUGDWDVXSSRUWWKHQRWLRQWKDWWXPRUFHOOVDWWKH

LQYDVLYHIURQWJUDGXDOO\FKDQJHWKHLUSKHQRW\SHWRIDFLOLWDWHLQYDVLRQLQWRVXUURXQGLQJWLVVXHV  

,QOLJKWRIWKHSLYRWDOUROHRI003LQFDQFHUSURJUHVVLRQWKHFXUUHQWUHSRUWSURYLGHV

VFLHQWLILF HYLGHQFH IRU LQGXFLQJ HQGRJHQRXV PL5DS H[SUHVVLRQ WR VXSSUHVV

003PHGLDWHG FDQFHU FHOO PLJUDWLRQ LQYDVLRQ DQG DQJLRJHQHVLV +RZHYHU EHFDXVH

PL5DS FDQ SRWHQWLDOO\ WDUJHW RYHU  GLIIHUHQW P51$V DV DQDO\]HG E\ 3LF7DU




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.




SUHGLFWLRQ DOJRULWKP  DQG PL5DSWDUJHWHG JHQHV PD\ H[HUW GLIIHUHQWLDO RU HYHQ

RSSRVLQJHIIHFWVLQGLIIHUHQWFHOOXODUFRQWH[WVWKHFKDOOHQJHDKHDGLVWRUHVROYHLQFRQVLVWHQW

REVHUYDWLRQVWKDWH[LVWDQGXQLI\WKHFXUUHQWGDWDLQWRDFRKHUHQWPHFKDQLVPIRUPL5DS

IXQFWLRQZLWKLQSDUWLFXODUWXPRUW\SHV,QWHUHVWLQJO\UHFHQWVWXGLHVXVLQJD'1$PLFURDUUD\

RU TXDQWLWDWLYH PDVV VSHFWURPHWU\ DSSURDFK LQGLFDWH WKDW WKH UHSUHVVLRQ RI P51$V DQG

SURWHLQV E\ D PL51$ LV RIWHQ UHODWLYHO\ VPDOO OHVV WKDQ  IROG DQG UDUHO\ H[FHHGV  IROG 

   *LYHQWKHWHFKQLFDOOLPLWDWLRQWKHRQO\ZD\WRYDOLGDWHWKHHIIHFWVRIUHGXFHGJHQH

H[SUHVVLRQ E\ D PL51$ RQ FHOOXODU SKHQRW\SLF FKDQJHV LV WR FKDUDFWHUL]H WKH

PL51$WDUJHWHG JHQH LQGLYLGXDOO\ 1HYHUWKHOHVV RXU ILQGLQJV LGHQWLI\ DQ DGGLWLRQDO

SRVWWUDQVFULSWLRQDO PHFKDQLVP IRU UHJXODWLQJ 003 H[SUHVVLRQ DQG LQGLFDWH WKDW

GRZQUHJXODWHG H[SUHVVLRQ RI PL5DS PD\ FRQWULEXWH WR FDQFHU LQYDVLRQ DQG

DQJLRJHQHVLVLQFHUWDLQWXPRUV  




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.




5HIHUHQFH
 .HVVHQEURFN.3ODNV9:HUE=0DWUL[PHWDOORSURWHLQDVHVUHJXODWRUVRIWKHWXPRUPLFURHQYLURQPHQW
&HOO  
 'XIRXU$6DPSVRQ16=XFNHU6&DR-5ROHRIWKHKHPRSH[LQGRPDLQRIPDWUL[PHWDOORSURWHLQDVHV
LQFHOOPLJUDWLRQ-&HOO3K\VLRO  
 &DR-.R]DUHNDU33DYODNL0&KLDUHOOL&%DKRX:)=XFNHU6'LVWLQFWUROHVIRUWKHFDWDO\WLFDQG
KHPRSH[LQ GRPDLQV RI PHPEUDQH W\SH PDWUL[ PHWDOORSURWHLQDVH LQ VXEVWUDWH GHJUDGDWLRQ DQG FHOO
PLJUDWLRQ-%LRO&KHP  
 6HLNL00HPEUDQHW\SHPDWUL[PHWDOORSURWHLQDVHDNH\HQ]\PHIRUWXPRULQYDVLRQ&DQFHUOHWWHUV
  
 &DR - &KLDUHOOL & 5LFKPDQ 2 =DUUDEL . .R]DUHNDU 3 =XFNHU 6 0HPEUDQH W\SH  PDWUL[
PHWDOORSURWHLQDVH LQGXFHV HSLWKHOLDOWRPHVHQFK\PDO WUDQVLWLRQ LQ SURVWDWH FDQFHU -%LRO&KHP
  
 /RKL-/HKWL.9DOWDQHQ+3DUNV:&.HVNL2MD-6WUXFWXUDODQDO\VLVDQGSURPRWHUFKDUDFWHUL]DWLRQ
RI WKH KXPDQ PHPEUDQHW\SH PDWUL[ PHWDOORSURWHLQDVH 07003  JHQH *HQH
  
 +DDV 7/ 6WLWHOPDQ ' 'DYLV 6- $SWH 66 0DGUL -$ (JU PHGLDWHV H[WUDFHOOXODU PDWUL[GULYHQ
WUDQVFULSWLRQRIPHPEUDQHW\SHPDWUL[PHWDOORSURWHLQDVHLQHQGRWKHOLXP7KH-RXUQDORIELRORJLFDO
FKHPLVWU\  
 3HWUHOOD%//RKL-%ULQFNHUKRII&(,GHQWLILFDWLRQRIPHPEUDQHW\SHPDWUL[PHWDOORSURWHLQDVHDVD
WDUJHW RI K\SR[LDLQGXFLEOH IDFWRU DOSKD LQ YRQ +LSSHO/LQGDX UHQDO FHOO FDUFLQRPD 2QFRJHQH
  
 =KDQJ+4L0/L64L70HL++XDQJ.HWDOPLFUR51$WDUJHWVPDWUL[PHWDOORSURWHLQDVHWR
LQKLELW LQYDVLRQ PHWDVWDVLV DQG DQJLRJHQHVLV RI QHXUREODVWRPD FHOOV 0ROHFXODU FDQFHU WKHUDSHXWLFV
  
 $NDQXPD 1 +RVKLQR , $NXWVX < 0XUDNDPL . ,VR]DNL < 0DUX\DPD 7 HW DO 0LFUR51$D
UHJXODWHVWKHP51$VRIWZRLQYDGRSRGLDUHODWHGSURWHLQV)6&1DQG003LQHVRSKDJHDOFDQFHU
%ULWLVKMRXUQDORIFDQFHU  
 0D/7HUX\D)HOGVWHLQ-:HLQEHUJ5$7XPRXULQYDVLRQDQGPHWDVWDVLVLQLWLDWHGE\PLFUR51$E
LQEUHDVWFDQFHU1DWXUH  
 %DUWHO'30LFUR51$VJHQRPLFVELRJHQHVLVPHFKDQLVPDQGIXQFWLRQ&HOO  
 *ULIILWKV-RQHV6*URFRFN5-YDQ'RQJHQ6%DWHPDQ$(QULJKW$-PL5%DVHPLFUR51$VHTXHQFHV
WDUJHWVDQGJHQHQRPHQFODWXUH1XFOHLFDFLGVUHVHDUFK 'DWDEDVHLVVXH '
 3DULNK$/HH&3HURQQH-0DUFKLQL6%DFFDULQL$.ROHY9HWDOPLFUR51$DKDVDFULWLFDOUROH
LQRYDULDQFDQFHUSURJUHVVLRQWKURXJKWKHUHJXODWLRQRIWKHHSLWKHOLDOPHVHQFK\PDOWUDQVLWLRQ1DWXUH
FRPPXQLFDWLRQV
 7D\ORU 0$ 6RVVH\$ODRXL . 7KRPSVRQ &/ 'DQLHOSRXU ' 6FKLHPDQQ :3 7*)EHWD XSUHJXODWHV
PL5D H[SUHVVLRQ WR SURPRWH EUHDVW FDQFHU PHWDVWDVLV 7KH -RXUQDO RI FOLQLFDO LQYHVWLJDWLRQ
  
 6KLQ.+%DH6'+RQJ+6.LP5+.DQJ0.3DUN1+PL5DVKRZVWXPRUVXSSUHVVLYHHIIHFW
DJDLQVW RUDO VTXDPRXV FHOO FDUFLQRPD FHOOV E\ GRZQUHJXODWLQJ .UDV %LRFKHPLFDO DQG ELRSK\VLFDO
UHVHDUFKFRPPXQLFDWLRQV  
 )HL - /L < =KX ; /XR ; PL5D SRVWWUDQVFULSWLRQDOO\ GRZQUHJXODWHV RQFRJHQLF 5DO$ DQG




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.



FRQWULEXWHV WR JURZWK LQKLELWLRQ DQG DSRSWRVLV LQ FKURQLF P\HORJHQRXV OHXNHPLD &0/  3OR6 RQH
  H
 6KL/&KHQJ==KDQJ-/L5=KDR3)X=HWDOKVDPLUDDQGKVDPLUEIXQFWLRQDVWXPRU
VXSSUHVVRUVLQKXPDQJOLRPDFHOOV%UDLQUHVHDUFK
 /L - =XFNHU 6 3XONRVNL*URVV $ .XVFX & .DUDD\YD] 0 -X - HW DO &RQYHUVLRQ RI VWDWLRQDU\ WR
LQYDVLYH WXPRU LQLWLDWLQJ FHOOV 7,&V  UROH RI K\SR[LD LQ PHPEUDQH W\SH PDWUL[ PHWDOORSURWHLQDVH
07003 WUDIILFNLQJ3OR6RQH  H
 (YHQVHQ 1$ .XVFX & 1JX\HQ +/ =DUUDEL . 'XIRXU $ .DGDP 3 HW DO 8QUDYHOLQJ WKH UROH RI
.,$$ D QRYHO HQGRSODVPLF UHWLFXOXP SURWHLQ LQ FDQFHU FHOO PLJUDWLRQ -RXUQDO RI WKH 1DWLRQDO
&DQFHU,QVWLWXWH  
 =XFNHU 6 3HL ' &DR - /RSH]2WLQ & 0HPEUDQH W\SHPDWUL[ PHWDOORSURWHLQDVHV 07003 
&XUU7RS'HY%LRO
 *LQRV0$3DJH*30LFKDORZLF]%63DWHO.-9RONHU6(3DPEXFFLDQ6(HWDO,GHQWLILFDWLRQRID
JHQHH[SUHVVLRQVLJQDWXUHDVVRFLDWHGZLWKUHFXUUHQWGLVHDVHLQVTXDPRXVFHOOFDUFLQRPDRIWKHKHDGDQG
QHFN&DQFHUUHVHDUFK  
 +DR < 7ULDGDILORSRXORV * 6DKEDLH3 <RXQJ +62PDU\0%/RZH$:*HQHH[SUHVVLRQ SURILOLQJ
UHYHDOV VWURPDO JHQHV H[SUHVVHG LQ FRPPRQ EHWZHHQ %DUUHWW
V HVRSKDJXV DQG DGHQRFDUFLQRPD
*DVWURHQWHURORJ\  
 5LFKDUGVRQ $/ :DQJ =& 'H 1LFROR $ /X ; %URZQ 0 0LURQ $ HW DO ; FKURPRVRPDO
DEQRUPDOLWLHVLQEDVDOOLNHKXPDQEUHDVWFDQFHU&DQFHU&HOO  
 6XQ/+XL$06X49RUWPH\HU$.RWOLDURY<3DVWRULQR6HWDO1HXURQDODQGJOLRPDGHULYHGVWHP
FHOOIDFWRULQGXFHVDQJLRJHQHVLVZLWKLQWKHEUDLQ&DQFHU&HOO  
 6DQFKH]&DUED\R 0 6RFFL 1' /R]DQR - 6DLQW ) &RUGRQ&DUGR & 'HILQLQJ PROHFXODU SURILOHV RI
SRRU RXWFRPH LQ SDWLHQWV ZLWK LQYDVLYH EODGGHU FDQFHU XVLQJ ROLJRQXFOHRWLGH PLFURDUUD\V -RXUQDO RI
FOLQLFDORQFRORJ\RIILFLDOMRXUQDORIWKH$PHULFDQ6RFLHW\RI&OLQLFDO2QFRORJ\  
 +HQGUL[ 1' :X 5 .XLFN 5 6FKZDUW] '5 )HDURQ (5 &KR .5 )LEUREODVW JURZWK IDFWRU  KDV
RQFRJHQLF DFWLYLW\ DQG LV D GRZQVWUHDP WDUJHW RI :QW VLJQDOLQJ LQ RYDULDQ HQGRPHWULRLG
DGHQRFDUFLQRPDV&DQFHUUHVHDUFK  
 +RQJ < 'RZQH\ 7 (X .: .RK 3. &KHDK 3< $
PHWDVWDVLVSURQH
 VLJQDWXUH IRU HDUO\VWDJH
PLVPDWFKUHSDLU SURILFLHQW VSRUDGLF FRORUHFWDO FDQFHU SDWLHQWV DQG LWV LPSOLFDWLRQV IRU SRVVLEOH
WKHUDSHXWLFV&OLQLFDO H[SHULPHQWDOPHWDVWDVLV  
 <X <3 /DQGVLWWHO ' -LQJ / 1HOVRQ - 5HQ % /LX / HW DO *HQH H[SUHVVLRQ DOWHUDWLRQV LQ SURVWDWH
FDQFHU SUHGLFWLQJ WXPRU DJJUHVVLRQ DQG SUHFHGLQJ GHYHORSPHQW RI PDOLJQDQF\ -RXUQDO RI FOLQLFDO
RQFRORJ\RIILFLDOMRXUQDORIWKH$PHULFDQ6RFLHW\RI&OLQLFDO2QFRORJ\  
 'HWZLOOHU .< )HUQDQGR 17 6HJDO 1+ 5\HRP 6: '
$PRUH 3$ <RRQ 66 $QDO\VLV RI
K\SR[LDUHODWHG JHQH H[SUHVVLRQ LQ VDUFRPDV DQG HIIHFW RI K\SR[LD RQ 51$ LQWHUIHUHQFH RI YDVFXODU
HQGRWKHOLDOFHOOJURZWKIDFWRU$&DQFHUUHVHDUFK  
 ,DFREX]LR'RQDKXH&$0DLWUD$2OVHQ0/RZH$:YDQ+HHN175RVW\&HWDO([SORUDWLRQRI
JOREDOJHQHH[SUHVVLRQSDWWHUQVLQSDQFUHDWLFDGHQRFDUFLQRPDXVLQJF'1$PLFURDUUD\V7KH$PHULFDQ
MRXUQDORISDWKRORJ\  
 .XVFX & (YHQVHQ 1 .LP ' +X <- =XFNHU 6 &DR - 7UDQVFULSWLRQDO DQG HSLJHQHWLF UHJXODWLRQ RI
.,$$JHQHH[SUHVVLRQLQKXPDQEUHDVWFDQFHU3OR6RQH  H
 &DR-'UHZV0/HH+0&RQQHU&%DKRX:)=XFNHU67KHSURSHSWLGHGRPDLQRIPHPEUDQHW\SH
 PDWUL[ PHWDOORSURWHLQDVH LV UHTXLUHG IRU ELQGLQJ RI WLVVXH LQKLELWRU RI PHWDOORSURWHLQDVHV DQG IRU




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.



DFWLYDWLRQRISURJHODWLQDVH$-%LRO&KHP  
 =XFNHU6&RQQHU&'L0DVVPR%,(QGH+'UHZV06HLNL0HWDO7KURPELQLQGXFHVWKHDFWLYDWLRQ
RISURJHODWLQDVH$LQYDVFXODUHQGRWKHOLDOFHOOV3K\VLRORJLFUHJXODWLRQRIDQJLRJHQHVLV7KH-RXUQDORI
ELRORJLFDOFKHPLVWU\  
 6DWR + .LQRVKLWD 7 7DNLQR 7 1DND\DPD . 6HLNL 0 $FWLYDWLRQ RI D UHFRPELQDQW PHPEUDQH W\SH
PDWUL[ PHWDOORSURWHLQDVH 07003  E\ IXULQ DQG LWV LQWHUDFWLRQ ZLWK WLVVXH LQKLELWRU RI
PHWDOORSURWHLQDVHV 7,03 )(%6/HWW  
 &DR - &KLDUHOOL & .R]DUHNDU 3 $GOHU +/ 0HPEUDQH W\SH PDWUL[ PHWDOORSURWHLQDVH SURPRWHV
KXPDQSURVWDWHFDQFHULQYDVLRQDQGPHWDVWDVLV7KURPE+DHPRVW  
 -LDQJ:*'DYLHV*0DUWLQ7$3DUU&:DWNLQV*0DVRQ0'HWDO([SUHVVLRQRIPHPEUDQHW\SH
PDWUL[PHWDOORSURWHLQDVH07003LQKXPDQEUHDVWFDQFHUDQGLWVLPSDFWRQLQYDVLYHQHVVRIEUHDVW
FDQFHUFHOOV,QW-0RO0HG  
 =DUUDEL . 'XIRXU $ /L - .XVFX & 3XONRVNL*URVV $ =KL - HW DO ,QKLELWLRQ RI PDWUL[
PHWDOORSURWHLQDVH  003 PHGLDWHG FDQFHU FHOO PLJUDWLRQ 7KH -RXUQDO RI ELRORJLFDO FKHPLVWU\
  
 (YHQVHQ 1$ /L - <DQJ - <X ; 6DPSVRQ 16 =XFNHU 6 HW DO 'HYHORSPHQW RI D KLJKWKURXJKSXW
WKUHHGLPHQVLRQDOLQYDVLRQDVVD\IRUDQWLFDQFHUGUXJGLVFRYHU\3OR6RQH  H
 /X & /L ;< +X < 5RZH 5* :HLVV 6- 07003 FRQWUROV KXPDQ PHVHQFK\PDO VWHP FHOO
WUDIILFNLQJDQGGLIIHUHQWLDWLRQ%ORRG  
 5LEDWWL ' 1LFR % 9DFFD $ 3UHVWD 0 7KH JHODWLQ VSRQJHFKRULRDOODQWRLF PHPEUDQH DVVD\ 1DWXUH
SURWRFROV  
 /H%OHX 96 0DFGRQDOG % .DOOXUL 5 6WUXFWXUH DQG IXQFWLRQ RI EDVHPHQW PHPEUDQHV ([SHULPHQWDO
ELRORJ\DQGPHGLFLQH  
 =KDQJ ; 1LH < /L ; :X * +XDQJ 4 &DR - HW DO 0LFUR51$D )XQFWLRQV DV DQ 2QFRPLU LQ
*DVWULF&DQFHUE\7DUJHWLQJWKH7XPRXU6XSSUHVVRU*HQH$703DWKRORJ\RQFRORJ\UHVHDUFK325

 -L'&KHQ=/L0=KDQ7<DR<=KDQJ=HWDO0LFUR51$DSURPRWHVWXPRUJURZWKDQGOLYHU
PHWDVWDVLVLQFRORUHFWDOFDQFHUE\WDUJHWLQJWKHWXPRUVXSSUHVVRU:,)0ROHFXODUFDQFHU
 .RUKDQ 3 (UGDO ( $WDEH\ 1 0L5DS LV GRZQUHJXODWHG LQ KHSDWRFHOOXODU FDUFLQRPD DQG
VXSSUHVVHV PRWLOLW\ LQYDVLRQ DQG EUDQFKLQJPRUSKRJHQHVLV E\ GLUHFWO\ WDUJHWLQJ F0HW %LRFKHPLFDO
DQGELRSK\VLFDOUHVHDUFKFRPPXQLFDWLRQV  
 0D / <RXQJ - 3UDEKDOD + 3DQ ( 0HVWGDJK 3 0XWK ' HW DO PL5 D 0<&0<&1DFWLYDWHG
PLFUR51$UHJXODWHV(FDGKHULQDQGFDQFHUPHWDVWDVLV1DW&HOO%LRO  
 $I]DO6/DODQL(13RXOVRP56WXEEV$5RZOLQVRQ*6DWR+HWDO07003DQG003P51$
H[SUHVVLRQ LQ KXPDQ RYDULDQ WXPRUV SRVVLEOH LPSOLFDWLRQV IRU WKH UROH RI GHVPRSODVWLF ILEUREODVWV
+XP3DWKRO  
 2KWDQL+0RWRKDVKL+6DWR+6HLNL01DJXUD+'XDORYHUH[SUHVVLRQSDWWHUQRIPHPEUDQHW\SH
PHWDOORSURWHLQDVHLQFDQFHUDQGVWURPDOFHOOVLQKXPDQJDVWURLQWHVWLQDOFDUFLQRPDUHYHDOHGE\LQVLWX
K\EULGL]DWLRQDQGLPPXQRHOHFWURQPLFURVFRS\,QWHUQDWLRQDOMRXUQDORIFDQFHU-RXUQDOLQWHUQDWLRQDOGX
FDQFHU  
 %DHN ' 9LOOHQ - 6KLQ & &DPDUJR )' *\JL 63 %DUWHO '3 7KH LPSDFW RI PLFUR51$V RQ SURWHLQ
RXWSXW1DWXUH  
 6HOEDFK06FKZDQKDXVVHU%7KLHUIHOGHU1)DQJ=.KDQLQ55DMHZVN\1:LGHVSUHDGFKDQJHVLQ
SURWHLQV\QWKHVLVLQGXFHGE\PLFUR51$V1DWXUH  




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.




/HJHQGV

)LJXUH8SUHJXODWLRQRI003LQLQYDVLYHKXPDQFDQFHUV$ '1$PLFURDUUD\GDWD

PLQLQJZDV SHUIRUPHGWR DQDO\]H WKH H[SUHVVLRQ RI003 LQ YDULRXV KXPDQ FDQFHUV% 

$QDO\VLVRI003P51$H[SUHVVLRQXVLQJUHDOWLPH573&5IRUWRWDO51$LVRODWHGIURP

DUFKLYHG ))3( WLVVXH VHFWLRQV RI '&,6 LQYDVLYH GXFWDO FDUFLQRPD DQG QRUPDO EUHDVW

HSLWKHOLDOFHOOVXVLQJD/&0WHFKQLTXH& 7LVVXHGLVWULEXWLRQRI003LQDGMDFHQWQRUPDO

EUHDVW HSLWKHOLXP DQG LQYDVLYH GXFWDO FDUFLQRPD H[DPLQHG E\ ,+& ZLWK DQ DQWL003

DQWLERG\ '(  ([DPLQDWLRQ RI 003 H[SUHVVLRQ LQ WXPRU DGMDFHQW QRUPDO WLVVXH

DUURZKHDG  DGHQRFDUFLQRPD RSHQ WULDQJOHV  DQG LQYDVLYH DGHQRFDUFLQRPD VWDUV  RI WKH

FRORQE\,+&ZLWKDQDQWL003DQWLERG\5HSUHVHQWDWLYHLPDJHVDUHVKRZQ (  

)LJXUH5HGXFHGH[SUHVVLRQRI003*)3ZLWKWKH
875LQOHVVLQYDVLYHFDQFHU

FHOOV $  0&) DQG 0'$0% FHOOV WUDQVIHFWHG ZLWK 003*)3


875 XSSHU

SDQHOV  DQG 003*)3 ZLWKRXW 


875 ORZHU SDQHOV  0LFURVFRSLF H[DPLQDWLRQ ZDV

SHUIRUPHG %  /1&D3 '8 DQG 3& FHOOV WUDQVIHFWHG ZLWK 003*)3


875 ZHUH

PLFURVFRSLFDOO\ H[DPLQHG &  &RPSXWDWLRQDO DQDO\VLV RI WKH PL51$ UHVSRQVH HOHPHQW

ZLWKLQWKH003
875UHYHDOHGDPL5ELQGLQJVLWH

)LJXUH  ,QWHUIHUHQFH ZLWK HFWRSLFDOO\ H[SUHVVHG 003 E\ PL5DS $ 

([SUHVVLRQ RI PL5DS DQG PL5 LQ &26 FHOOV WUDQVLHQWO\ H[SUHVVLQJ WKH

FRUUHVSRQGLQJ JHQHV ZDV H[DPLQHG E\ UHDOWLPH 573&5 /HYHOV RI H[SUHVVLRQ RI

PL51$VZHUHQRUPDOL]HGWR8VQ51$%& 7RWDO51$VDQGO\VDWHVLVRODWHGIURP&26

FHOOVH[SUHVVLQJF'1$VDVLQGLFDWHGZHUHH[DPLQHGIRU003H[SUHVVLRQXVLQJUHDOWLPH

573&5 +357DVDQRUPDOL]DWLRQFRQWURO  % DQG:HVWHUQEORWWLQJ WXEXOLQDVDORDGLQJ




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.




FRQWURO  &   '  1R HIIHFW RQ 003 H[SUHVVLRQ E\ PL5DS LQ &26 FHOOV

WUDQVIHFWHG ZLWK F'1$V HQFRGLQJ 003 ZLWKRXW WKH 


875 DV H[DPLQHG E\ :HVWHUQ

EORWWLQJ( $VFKHPDWLFGLDJUDPRIPL5DSVSRQJH) 0&)FHOOVVWDEO\H[SUHVVLQJ

WKHVSRQJHVDVLQGLFDWHGZHUHH[DPLQHGIRUPL5DSH[SUHVVLRQXVLQJUHDOWLPH573&5

* 0&)FHOOVVWDEO\H[SUHVVLQJWKHFRQWURODQGPL5DSVSRQJHVZHUHFXOWXUHGXQGHU

K\SR[LFFRQGLWLRQV 2 IRUKRXUVIROORZHGE\UHDOWLPH573&5IRU003+, 

0&) FHOOV VWDEO\ H[SUHVVLQJ WKH VSRQJHV ZHUH WUDQVIHFWHG ZLWK 003*)3
875

IROORZHGE\UHDOWLPH573&5IRU003 + DQG:HVWHUQEORWWLQJXVLQJDQDQWL003

DQWLERG\ , 

)LJXUH'LUHFWLQWHUDFWLRQRIPL51$DSZLWKWKH003
875$ $UHSRUWHU

JHQH DVVD\ ZDV SHUIRUPHG LQ +7 FHOOV WUDQVIHFWHG ZLWK F'1$V DV LQGLFDWHG 5HQLOOD

OXFLIHUDVHZDVXVHGDVDQRUPDOL]DWLRQFRQWURO% 0&)FHOOVVWDEO\H[SUHVVLQJWKHVSRQJHV

DV LQGLFDWHG ZHUH WUDQVIHFWHG ZLWK UHSRUWHU JHQHV DQG IROORZHG E\ D GXDO OXFLIHUDVH DVVD\

&'  0LFURVFRSLF H[DPLQDWLRQ RI WKH IOXRUHVFHQFH LQWHQVLW\ RI LQGLYLGXDO &26 FHOOV

FRWUDQVIHFWHG ZLWK 003*)3


875 F'1$V DORQJ ZLWK PL5FRQWURO RU PL5DS

7KH QXPEHU RI FHOOV GLVSOD\LQJ ZHDN IOXRUHVFHQW VLJQDO ZDV PLFURVFRSLFDOO\ FRXQWHG & 

5HSUHVHQWDWLYHLPDJHVDUHVKRZQ ' ( 0LFURVFRSLFH[DPLQDWLRQRIIOXRUHVFHQFHLQWHQVLW\

RILQGLYLGXDO0&)FHOOVFRH[SUHVVLQJ003*)3
875ZLWKVSRQJHV  

)LJXUH  (IIHFW RQ IXQFWLRQDO 003 E\ PL5DS $  7KH FRQGLWLRQHG PHGLXP

IURP &26 FHOOV WUDQVIHFWHG ZLWK D FRPELQDWLRQ RI '1$V DV LQGLFDWHG ZDV H[DPLQHG E\

JHODWLQ ]\PRJUDSK\ %  &RQGLWLRQHG PHGLXP DQG FHOO O\VDWHV ZHUH KDUYHVWHG IURP

&26FHOOVWUDQVIHFWHGZLWKYHFWRUFRQWURORU7,03F'1$DQG0'$0%FHOOVVWDEO\




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.




H[SUHVVLQJ PL5FRQWURO RU PL5DS DQG IROORZHG E\ :HVWHUQ EORWWLQJ XVLQJ DQ

DQWL7,03 DQWLERG\ WRS SDQHO  7KH PHPEUDQH ZDV VWULSSHG DQG WKHQ SUREHG ZLWK DQ

DQWLWXEXOLQDQWLERG\DVDORDGLQJFRQWURO ERWWRPSDQHO   &' &26FHOOVFRWUDQVIHFWHG

ZLWKF'1$VDVLQGLFDWHGZHUHH[DPLQHGE\DGRWEDVHGFHOOPLJUDWLRQDVVD\0LJUDWHGFHOOV

ZLWKLQ WKH 0LJUDWLRQ =RQH ZHUH PLFURVFRSLFDOO\ FRXQWHG XVLQJ WKH 1LNRQ 1,6 (OHPHQWV

VRIWZDUH & 5HSUHVHQWDWLYHLPDJHVDUHVKRZQ ' 

)LJXUH,QYHUVHFRUUHODWLRQEHWZHHQPL5DSDQG003LQKXPDQFDQFHUFHOO

OLQHV$% (OHYDWHG003H[SUHVVLRQLQLQYDVLYHFDQFHUFHOOOLQHVH[DPLQHGE\UHDOWLPH

573&5 $  DQG :HVWHUQ EORWWLQJ XVLQJ DQ DQWL003 DQWLERG\ %  &  5HGXFHG

PL5DSZDVREVHUYHGLQLQYDVLYHFDQFHUFHOOOLQHVDVH[DPLQHGE\UHDOWLPH573&5' 

PL5DS OHYHOV LQ WXPRU DGMDFHQW QRUPDO FHOOV DQG WXPRU FHOOV DW WKH LQYDVLYH IURQW

LVRODWHG IURP ))3( VHFWLRQV E\ D PDFURGLVVHFWLRQ WHFKQLTXH 5HDO WLPH 573&5 ZDV WKHQ

SHUIRUPHG XVLQJ WKH DSSURSULDWH SULPHUV ()  0'$0% FHOOV ZHUH WUDQVIHFWHG ZLWK

PL5FRQWURO DQG PL5DS IROORZHG E\ UHDO WLPH 573&5 WR H[DPLQH PL5DS

H[SUHVVLRQ (  7KH HIIHFW RQ 003 H[SUHVVLRQ LQ 0'$0% FHOOV H[SUHVVLQJ

PL5DSZDVH[DPLQHGE\UHDOWLPH573&5 )OHIWSDQHO RU:HVWHUQEORWWLQJ )ULJKW

SDQHO   *+  ([SUHVVLRQ RI PL5DS LQ +7 FHOOV ZDV H[DPLQHG E\ UHDO WLPH

573&5 *  DQG 003 H[SUHVVLRQ LQ WKH +7 FHOOV ZHUH H[DPLQHG E\ UHDO WLPH

573&5DQG:HVWHUQEORWWLQJ +  

)LJXUH  2YHUH[SUHVVLRQ RI PL5DS DWWHQXDWHV in vivo DQJLRJHQHVLV DQG LQYDVLRQ

E\FDQFHUFHOOV$ $WUDQVZHOOFKDPEHUPLJUDWLRQDVVD\ZDVSHUIRUPHGZLWK0'$0%

FHOOV VWDEO\ H[SUHVVLQJ PL5DS DQG PL5FRQWURO 0LJUDWHG FHOOV ZHUH DXWRPDWLFDOO\




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.




FRXQWHG EDVHG RQ QXFOHDU VWDLQLQJ /HIW SDQHO  5HSUHVHQWDWLYHLPDJHV DUH SUHVHQWHG 5LJKW

SDQHO  %  $ GRWEDVHG PLJUDWLRQ DVVD\ ZDV SHUIRUPHG ZLWK +7 FHOOV VWDEO\ LQIHFWHG

ZLWK UHWURYLUXV H[SUHVVLQJ PL51$ DV LQGLFDWHG &  $ ' LQYDVLRQ DVVD\ XVLQJ W\SH ,

FROODJHQ JHO ZDV SHUIRUPHG ZLWK 0'$0% FHOOV VWDEO\ H[SUHVVLQJ PL5DS

,QYDGHGFHOOVLQWKHLQYDVLRQ]RQHZHUHFRXQWHG /HIWSDQHO 5HSUHVHQWDWLYHLPDJHVRIFHOO

LQYDVLRQDUHVKRZQ ULJKWSDQHO ' 7KHHIIHFWRIPL5DSRQ+7FHOOLQYDVLRQZDV

H[DPLQHGXVLQJWKH'LQYDVLRQDVVD\( $FHOOVXUIDFHELRWLQ\ODWLRQDVVD\ZDVSHUIRUPHG

LQ +7 FHOOV VWDEO\ H[SUHVVLQJ PL5DS DQG FRQWURO DQG ZDV IROORZHG E\ :HVWHUQ

EORWWLQJ XVLQJ DQWL003 DQWLERG\  )  7KH &$0 LQYDVLRQ DVVD\ ZDV SHUIRUPHG ZLWK

0'$0% FHOOV VWDEO\ H[SUHVVLQJ PL5DS DQG FRQWURO )UR]HQ VHFWLRQV ZHUH

H[DPLQHG E\ ,+& XVLQJ DQ DQWL003 DQWLERG\ /HIW SDQHO  DQG + ( VWDLQLQJ 5LJKW

SDQHO  7XPRU FHOOV FDQ EH YLVXDOL]HG EDVHG RQ *)3 LQ WKH PL5DS RU PL5FRQWURO

SODVPLG'1$2SHQDUURZVXVHGIRUEORRGYHVVHOVDQGVROLGDUURZVXVHGIRUWXPRUFHOOVDW

WKHLQYDVLYHIURQW* &$0DQJLRJHQHVLVZDVSHUIRUPHGZLWK+7FHOOVRYHUH[SUHVVLQJ

PL5DS 1HZ EORRG YHVVHOV ZHUH FRXQWHG XQGHU D GLVVHFWLQJ PLFURVFRSH OHIW SDQHO 

5HSUHVHQWDWLYHLPDJHVRIQHZEORRGYHVVHOIRUPDWLRQDUHVKRZQ ULJKWSDQHO 




Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on May 14, 2015; DOI: 10.1158/0008-5472.CAN-14-2875
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

microRNA-181a-5p Inhibits Cancer Cell Migration and


Angiogenesis via Downregulation of Matrix
Metalloproteinase-14
Yiyi Li, Cem Kuscu, Anna Banach, et al.

Cancer Res Published OnlineFirst May 14, 2015.

Updated version Access the most recent version of this article at:
doi:10.1158/0008-5472.CAN-14-2875

Supplementary Access the most recent supplemental material at:


Material http://cancerres.aacrjournals.org/content/suppl/2015/05/15/0008-5472.CAN-14-2875.DC1.html

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been
Manuscript edited.

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Subscriptions Department at pubs@aacr.org.

Permissions To request permission to re-use all or part of this article, contact the AACR Publications
Department at permissions@aacr.org.

Downloaded from cancerres.aacrjournals.org on May 23, 2015. © 2015 American Association for Cancer Research.

You might also like