Tumor cell-driven extracellular matrix remodeling enables haptotaxis during metastatic progression Please share

advertisement
Tumor cell-driven extracellular matrix remodeling enables
haptotaxis during metastatic progression
The MIT Faculty has made this article openly available. Please share
how this access benefits you. Your story matters.
Citation
Oudin, M. J., O. Jonas, T. Kosciuk, L. C. Broye, B. C. Guido, J.
Wyckoff, D. Riquelme, et al. “Tumor Cell-Driven Extracellular
Matrix Remodeling Enables Haptotaxis During Metastatic
Progression.” Cancer Discovery (January 25, 2016).
As Published
http://dx.doi.org/10.1158/2159-8290.CD-15-1183
Publisher
American Association for Cancer Research
Version
Author's final manuscript
Accessed
Wed May 25 22:17:06 EDT 2016
Citable Link
http://hdl.handle.net/1721.1/101752
Terms of Use
Creative Commons Attribution-Noncommercial-Share Alike
Detailed Terms
http://creativecommons.org/licenses/by-nc-sa/4.0/
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
7XPRUFHOOGULYHQH[WUDFHOOXODUPDWUL[UHPRGHOLQJGULYHVKDSWRWD[LV
GXULQJPHWDVWDWLFSURJUHVVLRQ
0DGHOHLQH-2XGLQ2OLYHU-RQDV7DWVLDQD.RVFLXN/LOLDQH&%UR\H%UXQD&
*XLGR-HII:\FNRII'DLV\5LTXHOPH-RKQ0/DPDU6UHHMD%$VRNDQ&KDUOLH
:KLWWDNHU'XDQGXDQ0D5REHUW/DQJHU0LFKDHO-&LPD.DUL%:LVLQVNL5LFKDUG
2+\QHV'RXJODV$/DXIIHQEXUJHU3DWULFLD-.HHO\-DPHV(%HDU)UDQN%
*HUWOHU
'DYLG+.RFK,QVWLWXWHIRU,QWHJUDWLYH&DQFHU5HVHDUFK0,7&DPEULGJH0$86$
'HSDUWPHQWRI%LRORJLFDO(QJLQHHULQJ0,7&DPEULGJH0$86$
/LQHEHUJHU&RPSUHKHQVLYH&DQFHU&HQWHU81&&KDSHO+LOO&KDSHO+LOO1&86$
'HSDUWPHQWRI&HOODQG5HJHQHUDWLYH%LRORJ\8QLYHUVLW\RI:LVFRQVLQ0DGLVRQ
0DGLVRQ:,86$
'HSDUWPHQWRI%LRORJ\0,7&DPEULGJH0$86$
+RZDUG+XJKHV0HGLFDO,QVWLWXWH
'HSDUWPHQWRI0HGLFLQH8QLYHUVLW\RI:LVFRQVLQ&DUERQH&DQFHU&HQWHU0DGLVRQ
:,86$
5XQQLQJ7LWOH+DSWRWD[LVDQGPHWDVWDVLV
.H\ZRUGVEUHDVWFDQFHUFHOODGKHVLRQDQGH[WUDFHOOXODUPDWUL[FHOOPRWLOLW\DQG
PLJUDWLRQLPDJLQJRIWXPRUSURJUHVVLRQDQGPHWDVWDVLVPHWDVWDVLVPHWDVWDVLV
JHQHVPHWDVWDVLVPRGHOVWXPRUPDUNHUVDQGGHWHFWLRQRIPHWDVWDVLVKDSWRWD[LV0HQD
(QD9$63
$EEUHYLDWLRQVOLVW
&,FRQILGHQFHLQWHUYDO(&0H[WUDFHOOXODUPDWUL[(*)HSLGHUPDOJURZWKIDFWRU*)3
JUHHQIOXRUHVFHQWSURWHLQ)$.IRFDODGKHVLRQNLQDVH);IRFDOFRPSOH[HV)0,IRUZDUG
PLJUDWLRQLQGH[)1ILEURQHFWLQ,3LPPXQRSUHFLSLWDWLRQ+5KD]DUGUDWLR/1ODPLQLQ
003PDWUL[PHWDOORSURWHLQDVH57.UHFHSWRUW\URVLQHNLQDVH6+*VHFRQGKDUPRQLF
JHQHUDWLRQ7&*$7KH&DQFHU*HQRPH$WODV70$WLVVXHPLFURDUUD\91YLWURQHFWLQ
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
)LQDQFLDOVXSSRUW
7KLVZRUNZDVVXSSRUWHGE\D'R'%UHDVW&DQFHU5HVHDUFK3URJUDPSRVWGRFWRUDO
IHOORZVKLSWR:;:+WR0-2IXQGVIURPWKH/XGZLJ&HQWHUDW0,7WR)%*
DQG52+1,+JUDQW8&$WR)%*52+DQG'$/++0,IXQGLQJWR52+1,+
JUDQWV5&$5&$DQG8&$WR3-.D6FLHQFHZLWKRXW
ERUGHUV&13TIHOORZVKLSWR%&*WKH.RFK,QVWLWXWH)URQWLHU$ZDUGIURPWKH.DWK\DQG
.XUW0DUEOH5HVHDUFK)XQGWR)%*0-&DQG5/IXQGVIURPWKH3URVWDWH&DQFHU
)RXQGDWLRQWR2-DQG5/DQGWKH.RFK,QVWLWXWH1&,FRUHJUDQW3&$
&RUUHVSRQGLQJDXWKRU
)UDQN%*HUWOHU
.RFK,QVWLWXWHIRU,QWHJUDWLYH&DQFHU5HVHDUFK
0,7D
0DVVDFKXVHWWV$YH
&DPEULGJH0$
IJHUWOHU#PLWHGX
&RQIOLFWRILQWHUHVW)%*LVRQWKHDGYLVRU\ERDUGIRU0HWD6WDW0HWD6WDWGLGQRWSURYLGH
DQ\GLUHFWIXQGLQJIRUWKLVZRUN
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
$EVWUDFW
)LEURQHFWLQ)1LVDPDMRUFRPSRQHQWRIWKHWXPRUPLFURHQYLURQPHQWEXWLWVUROHLQ
SURPRWLQJPHWDVWDVLVLVLQFRPSOHWHO\XQGHUVWRRG+HUHZHVKRZWKDW)1JUDGLHQWVHOLFLW
GLUHFWLRQDOPRYHPHQWRIEUHDVWFDQFHUFHOOVin vitro DQGin vivo+DSWRWD[LVRQ)1
JUDGLHQWVUHTXLUHVGLUHFWLQWHUDFWLRQEHWZHHQĮȕLQWHJULQDQG0HQDDQDFWLQUHJXODWRU
DQGLQYROYHVLQFUHDVHVLQIRFDOFRPSOH[VLJQDOLQJDQGWXPRUFHOOPHGLDWHGH[WUDFHOOXODU
PDWUL[(&0UHPRGHOLQJ&RPSDUHGWR0HQDKLJKHUOHYHOVRIWKHSURPHWDVWDWLF
0HQD,19LVRIRUPDVVRFLDWHZLWKĮZKLFKHQDEOHV'KDSWRWD[LVRIWXPRUFHOOVWRZDUGV
WKHKLJK)1FRQFHQWUDWLRQVW\SLFDOO\SUHVHQWLQSHULYDVFXODUVSDFHDQGLQWKHSHULSKHU\
RIEUHDVWWXPRUWLVVXH0HQD,19DQG)1OHYHOVZHUHFRUUHODWHGLQWZREUHDVWFDQFHU
FRKRUWVDQGKLJKOHYHOVRI0HQD,19ZHUHVLJQLILFDQWO\DVVRFLDWHGZLWKLQFUHDVHGWXPRU
UHFXUUHQFHDVZHOODVGHFUHDVHGSDWLHQWVXUYLYDO2XUUHVXOWVLGHQWLI\DQRYHOWXPRUFHOO
LQWULQVLFPHFKDQLVPWKDWSURPRWHVPHWDVWDVLVWKURXJK(&0UHPRGHOLQJDQG(&0
JXLGHGGLUHFWLRQDOPLJUDWLRQ
6WDWHPHQWRIVLJQLILFDQFH
+HUHZHSURYLGHQHZLQVLJKWLQWRKRZWXPRUFHOO(&0LQWHUDFWLRQVJHQHUDWHVLJQDOVDQG
VWUXFWXUHVWKDWSURPRWHGLUHFWHGWXPRUFHOOPLJUDWLRQDFULWLFDOFRPSRQHQWRIPHWDVWDVLV
2XUUHVXOWVLGHQWLI\DWXPRUFHOOLQWULQVLFPHFKDQLVPGULYHQE\WKHDFWLQUHJXODWRU\
SURWHLQ0HQDWKDWSURPRWHV(&0UHPRGHOLQJDQGKDSWRWD[LVDORQJ)1JUDGLHQWV
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
,QWURGXFWLRQ
7KHWLVVXHPLFURHQYLURQPHQWLVFRPSRVHGRIVWURPDOFHOOVDQGH[WUDFHOOXODU
PDWUL[(&0DQGLVNQRZQWRFRQWULEXWHWRWXPRUSURJUHVVLRQ7KLVFRPSDUWPHQWLV
ULFKLQVXEVWUDWHERXQGDQGVROXEOHFXHVDQGSURYLGHVERWKWKHVWUXFWXUHDQGVLJQDOV
WKDWSURPRWHWXPRUFHOOSUROLIHUDWLRQVXUYLYDODQGLQYDVLRQ7KHPRVWDEXQGDQW(&0
SURWHLQVLQPRXVHPHWDVWDWLFEUHDVWWXPRUVDUHILEURQHFWLQ)1DQGFROODJHQV,Q
EUHDVWFDQFHUSDWLHQWVFROODJHQRUJDQL]DWLRQKDVKLJKSURJQRVWLFYDOXHDQG
LQFUHDVHG)1FRUUHODWHVZLWKGLVHDVHSURJUHVVLRQDQGPRUWDOLW\7KHFRQWH[WDQG
PHFKDQLVPVE\ZKLFKWXPRUFHOOVVHQVHDQGUHVSRQGWRFKDQJHVLQ(&0DEXQGDQFH
DQGDUFKLWHFWXUHGXULQJLQYDVLRQDQGPHWDVWDVLVKRZHYHUUHPDLQSRRUO\XQGHUVWRRG
&DQFHUFHOOVFDQUHVSRQGWRDYDULHW\RIFXHVLQRUGHUWRORFDOO\LQYDGHDQG
PHWDVWDVL]H*URZWKIDFWRUPHGLDWHGFKHPRWD[LVLVNQRZQWREHLPSRUWDQWIRUORFDO
LQYDVLRQDQGPHWDVWDVLV)DUOHVVLVNQRZQDERXWKDSWRWD[LVDSURFHVVLQZKLFKFHOO
PLJUDWLRQLVJXLGHGE\JUDGLHQWVRIVXUIDFHERXQGPROHFXOHVVXFKDV(&07KLVLV
SDUWLFXODUO\UHOHYDQWWRFDQFHUZKHUHWKHDPRXQWRI)1ZLWKLQWXPRUVFDQYDU\JUHDWO\
ZLWKKLJKFRQFHQWUDWLRQVRI)1W\SLFDOO\IRXQGQHDUEORRGYHVVHOVWXPRUSHULSKHU\DQGLQ
PHWDVWDWLFVLWHV:KLOH)1FDQDFWLYDWHLQWUDFHOOXODUVLJQDOLQJSDWKZD\VYLD
LQWHJULQVWKHSUHGRPLQDQWFODVVRIVXUIDFHDGKHVLRQUHFHSWRUVFHOOVFDQDOVR
UHPRGHOWKH(&0IRUH[DPSOHE\GULYLQJLQWHJULQPHGLDWHGDVVHPEO\RIVROXEOH)1LQWR
ILEULOV)1LVDOVRNQRZQWRSOD\DQLPSRUWDQWUROHLQFROODJHQILEULOORJHQHVLV%L
GLUHFWLRQDOFRPPXQLFDWLRQEHWZHHQFHOOVDQG(&0FXHVUHJXODWHVFHOOEHKDYLRUDVZHOO
DVWKHFRPSRVLWLRQDQGVWUXFWXUHRIWKHVXUURXQGLQJ(&0
0HQDDPHPEHURIWKH(QD9$63IDPLO\RIDFWLQILODPHQWHORQJDWLRQIDFWRUVLV
XSUHJXODWHGLQYDULRXVFDQFHUVDQGXQGHUJRHVDOWHUQDWLYHVSOLFLQJGXULQJEUHDVWFDQFHU
SURJUHVVLRQ0HQDELQGVWRWKH&WHUPLQDOHQGRIWKHF\WRSODVPLFWDLORIWKHLQWHJULQ
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
ĮYLDDQ/(5(5UHSHDWGRPDLQDEVHQWIURPRWKHU(QD9$63SURWHLQV,Q
ILEUREODVWV0HQDUHJXODWHVERWKRXWVLGHLQDQGLQVLGHRXWVLJQDOLQJDWIRFDOFRPSOH[HV
YLDLWVLQWHUDFWLRQZLWKĮ0HQD,19DQDOWHUQDWLYHO\VSOLFHGLVRIRUPFRQWDLQLQJD
DPLQRDFLGLQFOXVLRQHQFRGHGE\WKH³,19´H[RQLVH[SUHVVHGLQDJJUHVVLYHWXPRUFHOO
VXESRSXODWLRQV0HQD,19H[SUHVVLRQSURPRWHVPHWDVWDVLVE\LQFUHDVLQJVHQVLWLYLW\WR
(*)DQGWKHHIILFLHQF\RIPDWUL[GHJUDGDWLRQLQYDVLRQDQGLQWUDYDVDWLRQ
7RJHWKHUWKHVHILQGLQJVOHGXVWRK\SRWKHVL]HWKDWWKH(&0PD\DOVRSOD\DQLPSRUWDQW
UROHLQ0HQD0HQD,19GULYHQPHWDVWDVLV8VLQJSUHFOLQLFDOPRGHOVDQGDQDO\VLVRI
SDWLHQWVDPSOHVZHGHVFULEHDSUHYLRXVO\XQDSSUHFLDWHGPHFKDQLVPRIPHWDVWDVLV
ZKHUHXSUHJXODWLRQRI0HQDDQGLWVLQYDVLYHLVRIRUPHQGRZVWXPRUFHOOVZLWKWKHDELOLW\
WRPLJUDWHXS)1JUDGLHQWVDQGIDVKLRQWKHLURZQSDWKZD\WRZDUGVWKHEORRGVWUHDP
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
5HVXOWV
0HQDGULYHVKDSWRWD[LVRIWXPRUFHOOVRQ)1JUDGLHQWVYLDLWVLQWHUDFWLRQVZLWK
ĮȕDQG)DFWLQ
%DVHGRQWKHUHFHQWILQGLQJWKDW0HQDLQWHUDFWVZLWKWKHF\WRSODVPLFWDLORIRQHRI
WKHPDLQ)1UHFHSWRUVĮZHK\SRWKHVL]HGWKDW0HQDPD\EHLQYROYHGLQGLUHFWLRQDO
PLJUDWLRQUHVSRQVHVWRJUDGLHQWVRI)18VLQJDUHFHQWO\GHYHORSHGPLFURIOXLGLFGHYLFH
ZHVWXGLHGFHOOVPLJUDWLQJRQ)1JUDGLHQWVE\WLPHODSVHLPDJLQJDQGTXDQWLILHGWKHLU
IRUZDUGPLJUDWLRQLQGH[)0,WRDVVHVVKDSWRWD[LV)LJ$)LUVW09'
ILEUREODVWVZKLFKODFNDOOWKUHH(QD9$63SURWHLQV0HQD9$63DQG(9/PLJUDWHG
DFWLYHO\LQWKHGHYLFHKRZHYHUWKH\IDLOHGWRKDSWRWD[RQWKH)1JUDGLHQW,QWHUHVWLQJO\
0HQDEXWQRW9$63(9/H[SUHVVLQJ09'FHOOVH[KLELWHGDUREXVWKDSWRWDFWLF
UHVSRQVHDQGPLJUDWHGXSWKH)1JUDGLHQW)LJ%HYHQWKRXJKH[SUHVVLRQRIHDFKRI
WKHWKUHH(QD9$63SURWHLQVKDGSUHYLRXVO\UHSRUWHGVLPLODUHIIHFWVRQFHOOVSHHG
)LJ6$
:HQH[WH[DPLQHG0HQDGHSHQGHQWHIIHFWVRQKDSWRWD[LVRIEUHDVWFDQFHUFHOOV
,QVHUXPIUHHFRQGLWLRQV0'$0%FHOOVEHFRPHHQULFKHGRQWKH)1FRDWHG
XQGHUVLGHVRISRURXVILOWHUVLQWUDQVZHOODVVD\VKRZHYHUZHIRXQGWKDWWKLVFHOOW\SH
IDLOHGWRH[KLELWGLUHFWLRQDOPRYHPHQWRQ)1JUDGLHQWV)LJ&0'$0%FHOOOLQHV
VWDEO\H[SUHVVLQJ*)3WDJJHG0HQDRUFRQWURO*)3FRQVWUXFWDWOHYHOVVLPLODUWRWKRVH
VHHQLQLQYDGLQJFHOOVin vivoZHUHJHQHUDWHGUHIHUUHGWRDV&RQWURORU0HQD
)LJ6%&(FWRSLFH[SUHVVLRQRI0HQDHQDEOHGVLJQLILFDQWKDSWRWDFWLFUHVSRQVHV
RQ'JUDGLHQWVRI)1EXWQRWRIRQ'ODPLQLQ/1RUYLWURQHFWLQ91JUDGLHQWV
)LJ&ZLWKRXWDIIHFWLQJFHOOVSHHG)LJ6'9DU\LQJWKHFRQFHQWUDWLRQRIHLWKHU91RU
/1DIIHFWHGWKHVSHHGRI0'$0%DQG0HQDFHOOVEXWIDLOHGWRHOLFLWVLJQLILFDQW
KDSWRWDFWLFUHVSRQVHVDWDQ\FRQFHQWUDWLRQWHVWHG)LJ6'*
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
,Q'FROODJHQJHOVZLWK)1JUDGLHQWV0HQDH[SUHVVLRQDOVRLQGXFHGDVWURQJ
KDSWRWDFWLFUHVSRQVH)LJ'LQGHSHQGHQWO\RIYHORFLW\)LJ6(:KLOHWKHH[DFW
FRQFHQWUDWLRQRI)1LQWXPRUVLVXQNQRZQ)1LVH[SUHVVHGE\WXPRUDQGVWURPDOFHOOV
DQGDFFXPXODWHVLQWKHSHULYDVFXODUDUHDYLDOHDNDJHIURPWKHEORRGVWUHDPZKHUH)1
OHYHOVDVKLJKDVȝJPOKDYHEHHQREVHUYHG'XHWRWKHKHWHURJHQHRXVOHYHOVRI
)1IRXQGLQWXPRUVZHVWXGLHGKDSWRWD[LV'FROODJHQJHOVLQUHVSRQVHWRJUDGLHQWV
JHQHUDWHGIURPGLIIHUHQWVRXUFHFRQFHQWUDWLRQVRI)1,QKLJKOHYHOVRI)1XSWR
ȝJPO*)3DQG0HQDFHOOVZHUHXQDEOHWRPLJUDWHXSWKH)1JUDGLHQWDQG
LQVWHDGPLJUDWHGDZD\IURPWKH)1VRXUFHLQGLFDWLQJWKDWWKHSURKDSWRWDFWLFHIIHFWRI
0HQDRQ)1JUDGLHQWVLVFRQFHQWUDWLRQGHSHQGHQW
7KHUROHRILQWHJULQVLQ)1KDSWRWD[LVLQSDUWLFXODUWKHWZRPDMRU)1ELQGLQJ
LQWHJULQVĮȕDQGĮYȕLQWHJULQVUHPDLQVSRRUO\XQGHUVWRRG,QKLELWLRQRIĮȕE\WKH
IXQFWLRQEORFNLQJDQWLERG\3'EXWQRWRIĮYȕE\&LOHQJLWLGHEORFNHGKDSWRWD[LV
RI0HQDFHOOV)0,YDOXHVGHFUHDVHGE\RYHU)LJ(LQGLFDWLQJWKDW0HQD
GULYHQ)1KDSWRWD[LVUHTXLUHVĮȕVLJQDOLQJVSHFLILFDOO\:HWHVWHGZKHWKHU0HQD¶V
DELOLW\WRELQGĮYLDLWV/(5(5GRPDLQZDVUHTXLUHGIRU0HQDWRVXSSRUWKDSWRWD[LV
)LJ)0'$0%FHOOOLQHVVWDEO\H[SUHVVLQJ*)3WDJJHG0HQDLQZKLFKWKH/(5(5
GRPDLQZDVGHOHWHGWRDEURJDWHWKHLQWHUDFWLRQEHWZHHQ0HQDDQGĮ
0HQDǻ/(5(5VKRZHGQRDSSDUHQWGHIHFWVLQSURWHLQORFDOL]DWLRQDVMXGJHGE\WKH
*)3WDJFHOOPRUSKRORJ\FHOODUHDRUSUROLIHUDWLRQRQSODVWLFDWVWHDG\VWDWH
)LJ6%&)*0HQDǻ/(5(5FHOOVIDLOHGWRKDSWRWD[LQ'WR)1)0,VUHGXFHG
E\RYHU)LJ*KRZHYHUWKHLUPLJUDWLRQYHORFLW\ZDVVLPLODUWRFHOOVH[SUHVVLQJ
LQWDFW0HQD)LJ+6LPLODUUHVXOWVZHUHREWDLQHGLQ09'ILEUREODVWVRQD')1
JUDGLHQW)LJ6+,3UHYLRXVO\ZHIRXQGWKDWZKLOHWKH/(5(5GRPDLQZDVUHTXLUHG
IRUILEUREODVWVSUHDGLQJRQ)1WKH)DFWLQELQGLQJVLWHLQ0HQDZDV
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
GLVSHQVDEOH)LJ)7KHUHIRUHZHLQYHVWLJDWHGWKHUROHRIWKH)DFWLQELQGLQJ)$%
VLWHRI0HQDLQ)1GULYHQKDSWRWD[LV0HQDǻ)$%FHOOVIDLOHGWRKDSWRWD[LQD)1
JUDGLHQWLQD'FROODJHQJHO)LJ*ZKLOHDOVRGLVSOD\LQJVOLJKWUHGXFWLRQVLQFHOO
YHORFLW\)LJ+2YHUDOOWKHVHGDWDGHPRQVWUDWHWKDWVHQVLQJFKDQJHVLQ)1
FRQFHQWUDWLRQVGHSHQGVRQĮȕIXQFWLRQDVZHOODVWKHDELOLW\RI0HQDWRELQGĮDQG
WR)DFWLQ
0HQD,19GULYHVKDSWRWD[LVLQKLJK)1FRQFHQWUDWLRQVin vitroDQGin vivo
:HQH[WLQYHVWLJDWHGWKHUROHRIWKH0HQD,19LVRIRUPLQGULYLQJKDSWRWD[LV
&XOWXUHGFHOOVVKRZOLWWOHWRQRGHWHFWDEOH0HQD,19FRPSDUHGWRVSRQWDQHRXVRU
[HQRJUDIWPDPPDU\WXPRUV0'$0%DQG680FHOOOLQHVVWDEO\H[SUHVVLQJ
*)3WDJJHG0HQD,19RU0HQD,19ǻ/(5(5FRQVWUXFWVDWOHYHOVVLPLODUWRWKRVHJHQHUDWHG
IRU0HQDUHIHUUHGWRDVRU0HQD,19RU0HQD,19ǻ/(5(5ZHUHPDGH
)LJ6%&6XUSULVLQJO\XQOLNH0HQD0HQD,19HQDEOHGWXPRUFHOOKDSWRWD[LVWKURXJK
JUDGLHQWVRIKLJK)1FRQFHQWUDWLRQVHYHQXSWRȝJPO)LJ$HIIHFWVWKDWZHUH
LQGHSHQGHQWRIYHORFLW\)LJ6$%,QFRQWUDVWYDULHGFRQFHQWUDWLRQJUDGLHQWVRIHLWKHU
91RU/1IDLOHGWRHOLFLWVLJQLILFDQWKDSWRWDFWLFUHVSRQVHVE\0HQD,19FHOOV
VXJJHVWLQJWKLVUHVSRQVHZDVVSHFLILFWR)1)LJ6'(,QKLELWLRQRIĮȕEXWQRWRI
ĮYȕEORFNHGKDSWRWD[LVRI0HQD,19FHOOV)LJ%6&0HQD,19DOVRELQGVWKH
F\WRSODVPLFWDLORIĮGLUHFWO\YLDLWV/(5(5GRPDLQZHIRXQGWKDW0HQD,19ǻ/(5(5
DQG0HQD,19ǻ)$%FHOOVDOVRIDLOHGWRKDSWRWD[LQ'WR)1)LJ%7KHVHILQGLQJV
ZHUHFRQILUPHGLQ'DQG'FROODJHQJHOVXVLQJ680FHOOVH[SUHVVLQJWKHGLIIHUHQW
*)3WDJJHG0HQDLVRIRUPV)LJ6),2YHUDOOWKHVHGDWDVXJJHVWWKDWH[SUHVVLRQRI
0HQD,19HQDEOHVFHOOVWRKDSWRWD[DWKLJKHU)1FRQFHQWUDWLRQVWKDQ0HQD
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
:KLOHXSUHJXODWLRQRI)1LQDJJUHVVLYHWXPRUVLVWKRXJKWWRSURPRWHLQYDVLRQ
DQGPHWDVWDVLVZKHWKHU)1JUDGLHQWVSOD\DUROHLQJXLGLQJWXPRUFHOOPLJUDWLRQin vivo
KDVQRWEHHQHVWDEOLVKHG;HQRJUDIWWXPRUVZHUHJHQHUDWHGLQLQWKHPDPPDU\IDWSDG
RILPPXQRFRPSURPLVHGPLFHXVLQJ0'$0%DQG680FHOOV:HDVVD\HGWKH
DELOLW\RIFHOOVIURPWKHSULPDU\WXPRUWRLQYDGHDFWLYHO\LQWRPLFURQHHGOHVORDGHGZLWK
FROODJHQDQGLQFUHDVLQJFRQFHQWUDWLRQVRI)1&RQWUROWXPRUFHOOVZHUHQRW
DWWUDFWHGWR)1in vivo)LJ&ZKLOH0HQDWXPRUFHOOVH[KLELWHGDELSKDVLF
UHVSRQVHZLWKUREXVWLQYDVLRQE\0HQDFHOOVDWLQWHUPHGLDWH)1FRQFHQWUDWLRQVEXW
OLWWOHWRQRLQYDVLRQLQWRQHHGOHVZLWKHLWKHUORZRUKLJK)1FRQFHQWUDWLRQV)LJ&
,QWHUHVWLQJO\0HQD,19FHOOVZHUHVWLOODWWUDFWHGLQWRWKHQHHGOHVFRQWDLQLQJWKHKLJK
FRQFHQWUDWLRQVRI)1)LJ&:KLOH0HQDFDQSURPRWHLQYDVLRQin vivoLQUHVSRQVHWR
LQWHUPHGLDWH)1JUDGLHQWV0HQD,19DOORZVWXPRUFHOOVWRPLJUDWHWKURXJKVXEVWDQWLDOO\
KLJKHUIROGJUHDWHU)1FRQFHQWUDWLRQV
7RYLVXDOL]H)1GULYHQKDSWRWDFWLFUHVSRQVHVLQVLGHWXPRUVZHXVHGD
PLFURVFDOHLPSODQWDEOHGHYLFHWKDWDOORZVIRUin vivoUHOHDVHRIPROHFXOHVLQ
JUDGLHQWV'HYLFHVILOOHGZLWK5KRGDPLQHODEHOHG)1ZHUHLPSODQWHGQHDUWKHHGJHV
RI0'$0%RU680RUWKRWRSLFWXPRUVWRJHQHUDWHKLJKFRQFHQWUDWLRQ)1
JUDGLHQWV)LJ'8VLQJLQWUDYLWDOLPDJLQJFHOOPRWLOLW\DQG)0,VZHUHTXDQWLILHGLQ
UHVSRQVHWRJUDGLHQWVRI)1RUWRIOXRUHVFHQWO\ODEHOHGGH[WUDQVLPLODURIVL]HWR)1DVD
FRQWURO)LJ6-([SUHVVLRQRI0HQD,19EXWQRW0HQDLQKXPDQ0'$0%DQG
680FHOOVVLJQLILFDQWO\LQFUHDVHGWKHQXPEHURIFHOOVPRYLQJLQWKH[HQRJUDIWWXPRUV
DQHIIHFWGHSHQGHQWRQWKHLQWHUDFWLRQZLWKĮȕ)LJ6/0,QWXPRUVLPSODQWHGZLWK
)1ORDGHGGHYLFHV0HQD,19EXWQRW&RQWURORU0HQDFHOOVPRYHGWRZDUGV
WKH)1JUDGLHQW)LJ(UHSUHVHQWDWLYHLPDJHVZLWKFHOOWUDFNV)LJ6.DQG
VXSSOHPHQWDU\YLGHRV,QFRQWUDVW0HQDǻ/(5(5DQG0HQD,19ǻ/(5(5FHOOV
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
GLGQRW6LPLODUO\FHOOVLQ0HQD,19WXPRUVPLJUDWHGWRZDUGVWKH)1DQHIIHFWWKDW
ZDVDEVHQWLQ&RQWURORU0HQD,19ǻ/(5(5WXPRUFHOOV)LJ)$OWRJHWKHU
XVLQJWKHQHHGOHFROOHFWLRQDVVD\DQGLQWUDYLWDOLPDJLQJZHVKRZIRUWKHILUVWWLPHWKDW
KDSWRWD[LVWRZDUGV)1RFFXUULQJin vivoLVGULYHQE\0HQD,19DQGLWVLQWHUDFWLRQZLWKĮ
0HQDLVRIRUPH[SUHVVLRQFRUUHODWHVZLWK)1DQGLQWHJULQĮH[SUHVVLRQOHYHOVDV
ZHOODVRXWFRPHLQKXPDQEUHDVWFDQFHUSDWLHQWV
3UHYLRXVZRUNGHPRQVWUDWHGWKDWIRUFHGH[SUHVVLRQRI0HQD,19GULYHVPHWDVWDVLV
LQ[HQRJUDIWWXPRUPRGHOVDQGWKDW0HQD,19P51$OHYHOVDVGHWHFWHGE\T3&5DUH
UHODWLYHO\KLJKHULQFHOOVWKDWLQWUDYDVDWHHIILFLHQWO\DQGLQSDWLHQWVZLWKKLJKQXPEHUVRI
70(0DVWUXFWXUHFRQWDLQLQJDWXPRUFHOOPDFURSKDJHDQGHQGRWKHOLDOFHOODVVRFLDWHG
ZLWKWKHOLNHOLKRRGRIPHWDVWDVLVLQ(5+HUEUHDVWFDQFHUSDWLHQWV+RZHYHUWKH
UHODWLRQVKLSEHWZHHQ0HQD,19P51$RUSURWHLQOHYHOVDQGSDWLHQWRXWFRPHLQKXPDQ
EUHDVWFDQFHUSDWLHQWVKDVQRWEHHQLQYHVWLJDWHG)LUVWZHDQDO\]HGWKHEUHDVW
FDQFHUSDWLHQWVLQWKH7&*$FRKRUWZLWK51$VHTDQGFOLQLFDOGDWDDYDLODEOH6LQFH
WKH,19H[RQZDVQRWDQQRWDWHGZKHQWKH51$VHTGDWDZHUHILUVWDQDO\]HGZH
DFFHVVHGWKHUDZVHTXHQFHGDWDDQGPDSSHGUHDGVLQHDFKVDPSOHWRDOO0HQDH[RQV
6HSDUDWLQJSDWLHQWVLQWRTXDUWLOHVDFFRUGLQJWR0HQDH[SUHVVLRQIDLOHGWRUHYHDODQ\
VLJQLILFDQWFRUUHODWLRQVEHWZHHQ0HQDOHYHOVMXGJHGE\OHYHOVRIFRQVWLWXWLYHO\LQFOXGHG
H[RQVDQGRYHUDOOVXUYLYDOLQWKHHQWLUH7&*$EUHDVWFDQFHUFRKRUW)LJ6$RULQWKH
VXEVHWRISDWLHQWVZLWK!\UIROORZXS)LJ$+RZHYHUSDWLHQWVZLWKKLJKOHYHOVWRS
RI0HQD,19P51$DVDVVHVVHGE\WKHDEXQGDQFHRI,19H[RQVHTXHQFHUHDGV
H[KLELWHGVLJQLILFDQWO\UHGXFHGVXUYLYDOFRPSDUHGWRSDWLHQWVLQHDFKRIWKHWKUHHORZHU
TXDUWLOHVRI0HQD,19H[SUHVVLRQ)LJV%6%6LPLODUUHVXOWVZHUHIRXQGLQWKHQRGH
QHJDWLYHSDWLHQWVXEJURXS)LJ6()XUWKHUPRUHERWK&R[DQGORJLVWLFUHJUHVVLRQ
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
GHPRQVWUDWHGWKDW0HQD,19ZDVDVXEVWDQWLDOO\VWURQJHUSUHGLFWRURISRRURXWFRPHLQ
SDWLHQWVZLWK\HDUIROORZXSWKDQ0HQDDORQH)LJV&'6&'PRGHOVFRPELQLQJ
0HQD,19DQG0HQDH[SUHVVLRQOHYHOVIDLOHGWRLQFUHDVHWKHSUHGLFWLYHSRZHUEH\RQGWKDW
RI0HQD,19:HQH[WVWXGLHGKRZ0HQD,19OHYHOVFRUUHODWHGZLWK)1DQGĮH[SUHVVLRQLQ
WKLVGDWDVHW2YHUDOO0HQDDQG0HQD,19H[SUHVVLRQZHUHERWKVLJQLILFDQWO\FRUUHODWHG
ZLWK)1DQGWRDOHVVHUGHJUHHĮ)LJ6),QSDUWLFXODULQSDWLHQWVZLWK!\UIROORZ
XSZHREVHUYHGDKLJKO\VLJQLILFDQWFRUUHODWLRQEHWZHHQ0HQD0HQD,19DQG)1RUĮLQ
SDWLHQWVWKDWVXFFXPEHGWRWKHLUGLVHDVHWKDWZDVDEVHQWLQVXUYLYLQJSDWLHQWV
)LJ6*+
8VLQJDQHZO\GHYHORSHGDQWLERG\VSHFLILFIRU0HQD,19ZHWKHQLQYHVWLJDWHGWKH
UHODWLRQVKLSEHWZHHQHQGRJHQRXV0HQD,19ĮDQG)1SURWHLQXVLQJLPPXQRVWDLQLQJLQ
WKH00793\07VSRQWDQHRXVPRXVHPRGHORIEUHDVWFDQFHUDQGLQDSUHYLRXVO\
FKDUDFWHUL]HGWLVVXHPLFURDUUD\70$RISDWLHQWV%RWK0HQDDQG0HQD,19DUH
H[SUHVVHGLQ3\07WXPRUV)LJ(DQGFDQEHGHWHFWHGLQFHOOVWKDWDOVRH[SUHVVĮȕ
)LJ)0HQD,19H[SUHVVLRQDQGGLVWULEXWLRQVLJQLILFDQWO\FRUUHODWHGZLWKWKDWRI)1LQ
WKLVPRGHO)LJ*+:HDOVRIRXQGDVLJQLILFDQWFRUUHODWLRQEHWZHHQ)1DQG0HQD,19
OHYHOVLQWKH70$)LJ,-6LPLODUO\LQSDWLHQWVUHSUHVHQWHGE\WKH70$KLJKHU0HQD,19
OHYHOVZHUHVLJQLILFDQWO\FRUUHODWHGZLWKSRRURXWFRPH)LJ6,,QDGGLWLRQSDWLHQWVZLWK
UHFXUUHQWGLVHDVHHLWKHUORFDORUDWDGLVWDQWVLWHKDGVLJQLILFDQWO\KLJKHUOHYHOVRI
0HQD,19)LJ./RJLVWLFUHJUHVVLRQDQDO\VLVLQGLFDWHGWKDW0HQD,19H[SUHVVLRQ0HQD,19
ZDVDVLJQLILFDQWSUHGLFWRURIUHFXUUHQFH&RHIILFLHQWRIS $QDYHUDJH
IROGLQFUHDVHLQ0HQD,19H[SUHVVLRQFRUUHODWHGZLWKDIROGLQFUHDVHLQWKHQXPEHU
RISDWLHQWVZLWKUHFXUUHQFH)LJ6-.)XUWKHULQFUHDVHVLQ0HQD,19H[SUHVVLRQGLGQRW
FRUUHODWHZLWKIXUWKHULQFUHDVHVLQUHFXUUHQFHVXJJHVWLQJWKDWHYHQVPDOOLQFUHDVHVLQ
0HQD,19SURWHLQH[SUHVVLRQFDQDIIHFWUHFXUUHQFH:KLOHP51$OHYHOVRIHLWKHU0HQD,19
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
RU)1DORQHGLGQRWFRUUHODWHZLWKWLPHWRGLVHDVHUHFXUUHQFHSDWLHQWVZLWKKLJKOHYHOVRI
both0HQD,19DQG)1VKRZHGDVWDWLVWLFDOO\VLJQLILFDQWGHFUHDVHLQWLPHWRUHFXUUHQFH
)LJ/7RJHWKHUWKHVHGDWDSURYLGHWKHILUVWHYLGHQFHWKDW0HQD,1951$DQGSURWHLQ
OHYHOVFRUUHODWHZLWKWXPRUUHFXUUHQFHDQGVXUYLYDODQGVXSSRUWDOLQNEHWZHHQ
HQGRJHQRXV0HQD,19ĮDQG)1H[SUHVVLRQLQEUHDVWFDQFHUSDWLHQWV
7RHYDOXDWHWKHUROHRI0HQD0HQD,19GULYHQKDSWRWD[LVLQPHWDVWDVLVZH
TXDQWLILHGWKHQXPEHURIVSRQWDQHRXVPHWDVWDVHVLQWKHOXQJVRIPLFHZLWK0'$0%
DQG680[HQRJUDIWV0LFHZHUHVDFULILFHGZKHQSULPDU\WXPRUVL]HUHDFKHGFPLQ
GLDPHWHU,QWHUHVWLQJO\WKHLQLWLDOJURZWKUDWHRIDQG0HQD,19ǻ/(5(5WXPRUV
ZDVVORZHUWKDQWKDWRIWXPRUVJHQHUDWHGIURPWKHRWKHUFHOOOLQHV)LJ6$%:KLOH
WKHUHZHUHRQO\PLQRUFKDQJHVLQSUROLIHUDWLRQOHYHOVDVGHWHFWHGXVLQJWKHPDUNHU.L
0HQD,19ǻ/(5(5WXPRUVKDGDIROGLQFUHDVHLQOHYHOVRIWKHDSRSWRWLFPDUNHU
FOHDYHGFDVSDVH)LJ6'(7KLVGHOD\LQWXPRUJURZWKDQGLQFUHDVHLQFHOOGHDWK
FRXOGEHUHVFXHGE\FRLQMHFWLQJ0HQD,19ǻ/(5(5FHOOVZLWKZLOGW\SH0'$0%
FHOOVH[SUHVVLQJP&KHUU\RUE\OHWWLQJWKHPJURZIRUZHHNVLQVWHDGRIWRUHDFKFP
LQGLDPHWHU)LJ6'(([SUHVVLRQRI0HQD,19VLJQLILFDQWO\LQFUHDVHGWKHPHWDVWDWLF
LQGH[LQWKHOXQJVIROGLQERWKKXPDQFHOOOLQHVDWZHHNVFRPSDUHGWRFRQWUROV
)LJ02'HOHWLRQRIWKH/(5(5UHJLRQLQERWK0HQDDQG0HQD,19VLJQLILFDQWO\
GHFUHDVHGWKHOXQJPHWDVWDWLFLQGH[)LJ126LPLODUUHVXOWVZHUHIRXQGLQWKHSRRUO\
PHWDVWDWLF680FHOOOLQH)LJ6(6LPLODUO\GHSOHWLRQRIDOO0HQDLVRIRUPVLQ
0'$0%FHOOVXVLQJVK51$DOVRGHOD\HGWXPRUJURZWKDVZHOODVGHFUHDVLQJWXPRU
FHOOPRWLOLW\DQGPHWDVWDVLV)LJ6*07KHVHGDWDVXSSRUWDPRGHOZKHUHE\
0HQD0HQD,19GULYHQĮGHSHQGHQWKDSWRWD[LVSOD\VDNH\UROHLQSURPRWLQJWXPRU
PHWDVWDVLV
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
+DSWRWD[LVRQ)1JUDGLHQWVLVGHSHQGHQWRQVLJQDOLQJDWIRFDOFRPSOH[HV);V
:HQH[WLQYHVWLJDWHGWKHPHFKDQLVPE\ZKLFK0HQDLVRIRUPVGULYHKDSWRWD[LVDW
ORZDQGKLJK)1FRQFHQWUDWLRQV2QHZD\)1FDQVXSSRUWFHOOPLJUDWLRQLVE\DFWLYDWLQJ
LQWHJULQPHGLDWHGLQWUDFHOOXODUVLJQDOLQJSDWKZD\VDW);VWKDWSURPRWHFHOOPRWLOLW\
:KHQSODWHGRQ'FROODJHQDQG)1ERWK0HQDDQG0HQD,19FHOOVVKRZHG
LQFUHDVHVLQFHOODUHDDQGLQWKHQXPEHURIĮSRVLWLYHDGKHVLRQVUHODWLYHWRFRQWUROFHOOV
RUWRFHOOVSODWHGRQFROODJHQRQO\DQHIIHFWGHSHQGHQWRQĮDFWLYLW\DVZHOODVWKHĮ
ELQGLQJ/(5(5GRPDLQ)LJ$(:KLOH0HQD,19FHOOVVKRZHGDLQFUHDVHLQ
ERWKWRWDODQGVXUIDFHOHYHOVRIĮWKHVWHDG\VWDWHOHYHOVRIĮYȕDQGRWKHULQWHJULQV
H[DPLQHGZHUHXQFKDQJHG)LJ6$*)XUWKHUPRUHSODWLQJFHOOVRQ)1DOVRLQFUHDVHG
WKHOHYHOVRIĮZLWKLQ0HQDSRVLWLYHDGKHVLRQVLQGLFDWHGE\WKH*)3WDJZLWK
0HQD,19FHOOVVKRZLQJLQFUHDVHGĮOHYHOVUHODWLYH0HQDFHOOV)LJ%)7KH)1
GULYHQUHFUXLWPHQWRIĮWR0HQDSRVLWLYHDGKHVLRQVZDVGHSHQGHQWRQĮDFWLYLW\DV
ZHOODVWKH/(5(5GRPDLQIRUERWKLVRIRUPV)LJ)*0HQD,19ǻ/(5(5FHOOVDOVR
KDGUHGXFHGQXPEHURI0HQDSRVLWLYHDGKHVLRQV)LJ6,
3KRVSKRU\ODWLRQRIVHYHUDOSURWHLQVDWIRFDODGKHVLRQVGRZQVWUHDPRILQWHJULQ
DFWLYDWLRQSDUWLFXODUO\IRFDODGKHVLRQNLQDVH)$.LVLPSRUWDQWIRUPRWLOLW\DQG
WXPRULJHQHVLV3KDUPDFRORJLFDOLQKLELWLRQRI)$.LQKLELWHG0HQDDQG0HQD,19
GULYHQKDSWRWD[LVRQORZ)1JUDGLHQWV)LJ+,PPXQRIOXRUHVFHQFHRIKDSWRWD[LQJFHOOV
UHYHDOHGWKDWERWK0HQDDQG0HQD,19FHOOVVKRZLQFUHDVHGQXPEHURIS)$.
SRVLWLYHDGKHVLRQV)LJ,-,Q0HQD,19ǻ/(5(5RU0HQD,19ǻ)$%FHOOVWKH
QXPEHURIS)$.SRVLWLYHDGKHVLRQVLQWKHFHOOVZDVVLJQLILFDQWO\ORZHUWKDQLQ
0HQD,19FHOOV)LJ.:KHQFHOOVZHUHSODWHGRQKLJK)1JUDGLHQWVRQO\0HQD,19
FHOOVKDSWRWD[HG)LJ6-DQGDFFRUGLQJO\WKHVHFHOOVKDGDVLJQLILFDQWLQFUHDVHLQWKH
QXPEHURIS)$.SRVLWLYHDGKHVLRQVUHODWLYHWR0HQDRUFRQWUROFHOOV)LJ/
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
7KHVHGDWDVXJJHVWWKDWRXWVLGHLQDFWLYDWLRQRILQWHJULQVDQG0HQDGHSHQGHQW);
VLJQDOLQJE\)1LVLPSRUWDQWIRUKDSWRWD[LVRIWXPRUFHOOV
7KHGLIIHUHQFHVEHWZHHQIRFDOFRPSOH[FRPSRVLWLRQVLJQDOLQJDQGKDSWRWD[LV
DULVLQJIURPH[SUHVVLRQRI0HQD,19YV0HQDSURPSWHGXVWRDVNZKHWKHUZHFRXOG
GHWHFWDQ\UHOHYDQWELRFKHPLFDOGLIIHUHQFHVEHWZHHQWKHLVRIRUPV3UHYLRXVO\ZH
GHPRQVWUDWHGWKDW0HQDFRXOGEHGHWHFWHGLQFRPSOH[ZLWKĮE\FR
LPPXQRSUHFLSLWLDWLRQ,3:HSHUIRUPHGĮ,3VWRFRPSDUHWKHDPRXQWVRI0HQD
DQG0HQD,19LQFRPSOH[ZLWKĮDQGUHSURGXFLEO\GHWHFWHGDQDYHUDJHIROGJUHDWHU
OHYHORI0HQD,19FRPSDUHGWR0HQDE\ZHVWHUQEORWRIDQWLĮ,3V)LJ017KXV
LQFOXVLRQRI,19LQ0HQDOHDGVWRVLJQLILFDQWO\LQFUHDVHGDVVRFLDWLRQZLWKĮȕ
0HQD,19GULYHQ(&0UHPRGHOLQJLVUHTXLUHGIRU'KDSWRWD[LV
)1FRXOGDOVRVXSSRUWKDSWRWD[LVWKURXJKLWVUROHLQSURYLGLQJVWUXFWXUDOVXSSRUW
IRUFHOOVSDUWLFXODUO\LQ'DQGLQWKHSUHVHQFHRIFROODJHQ)1ILEULOORJHQHVLVH[SRVHV
FU\SWLFELQGLQJVLWHVLQ)1QRUPDOO\KLGGHQLQWKHJOREXODUIRUPDQGILEULOODU)1FDQ
ELQGFROODJHQDQGUHJXODWHLWVGHSRVLWLRQDQGRUJDQL]DWLRQ0HQDDQG0HQD,19
FHOOVKDGLQFUHDVHGQXPEHURISURWUXVLRQVLQORZ)1JUDGLHQWVZKLOHLQKLJK)1
JUDGLHQWVRQO\0HQD,19FHOOVVKRZHGWKLVSKHQRW\SH)LJ6$6LPLODUO\
0HQD,19FHOOVZHUHPRUHHORQJDWHGWKDQ0HQDRU&RQWUROFHOOVLQWKHSUHVHQFHRID)1
JUDGLHQWin vivoDQHIIHFWGHSHQGHQWRQWKHLQWHUDFWLRQZLWKĮ)LJ6%&:HDOVR
IRXQGWKDW0HQD,19H[KLELWHGLQFUHDVHGDFFXPXODWLRQRI)1)LJ$%DQG
FROODJHQ)LJ$&DWERWKORZDQGKLJK)1FRQFHQWUDWLRQVDIWHUKUVZKLOHQHLWKHU
0HQDQRU&RQWUROFHOOVH[KLELWHGDQ\VLJQLILFDQWDFFXPXODWLRQRIWKHWZR(&0
SURWHLQV)1WULJJHUHGRXWVLGHLQVLJQDOLQJZDVLQVXIILFLHQWWRVXSSRUW0HQD,19GHSHQGHQW
KDSWRWD[LVLQ'JHOVDV0HQD,19FHOOVIDLOHGWRKDSWRWD[RQDJUDGLHQWRI)1D
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
IUDJPHQWRI)1WKDWDFWLYDWHVVLJQDOLQJYLD5*'ELQGLQJLQWHJULQVEXWFDQQRWIRUPILEULOV
)LJ',QKLELWLRQRIILEULOORJHQHVLVE\DGGLWLRQRIDN')1IUDJPHQWWKDWFRQWDLQV
WKHFU\SWLF)1DQGFROODJHQELQGLQJVLWHVLQ)1EORFNHG0HQD,19GULYHQ'
KDSWRWD[LV)LJ(7KHUHIRUHWKHIRUPDWLRQRI)1ILEULOVLVLPSRUWDQWIRU)1WRHYRNH
GLUHFWLRQDOPRWLOLW\RIWXPRUFHOOVLQ'$GGLWLRQRIWKHN')1IUDJPHQWWRVSUHDGLQJ
0HQD,19FHOOVGHFUHDVHGWKHQXPEHURIĮSRVLWLYHDGKHVLRQVDVZHOODVĮ
UHFUXLWPHQWWR0HQD,19SRVLWLYHDGKHVLRQV)LJ6'*VXJJHVWLQJWKDWILEULOODU)1PD\
HQKDQFHDVVHPEO\RIĮȕFRQWDLQLQJDGKHVLRQV
,Q0HQD,19FHOOVLQKLELWLRQRIĮȕGHFUHDVHGDFFXPXODWLRQRIERWK)1DQG
FROODJHQZKLOHLQKLELWLRQRIĮYȕKDGOLWWOHRUQRHIIHFW0HQD,19ǻ/(5(5DQG
0HQD,19ǻ)$%FHOOVKDGGHFUHDVHG)1DQGFROODJHQ)LJ)*DFFXPXODWLRQDURXQGWKH
FHOOV,QKLELWLRQRISURWHDVHDFWLYLW\ZLWKWKHEURDGVSHFWUXPPDWUL[PHWDOORSURWHLQDVH
003LQKLELWRU%%KDGQRHIIHFWRQ(&0UHRUJDQL]DWLRQEXWLQKLELWLRQRIDFWRP\RVLQ
FRQWUDFWLOLW\E\WKH52&.LQKLELWRU<GLGGHFUHDVHFROODJHQDQG)1DFFXPXODWLRQ
DURXQGWKHFHOOV)LJ6+,7KHVHUHVXOWVLQGLFDWHWKDW0HQD,19GULYHQĮGHSHQGHQW)1
DQGFROODJHQDFFXPXODWLRQE\WKHWXPRUFHOOVWKHPVHOYHVLVLPSRUWDQWIRUKDSWRWD[LV
0HQD,19H[SUHVVLRQGULYHVFROODJHQUHRUJDQL]DWLRQLQWXPRUV
*LYHQWKHUROHRI0HQD,19LQGULYLQJ(&0UHRUJDQL]DWLRQin vitroWKHLPSRUWDQFH
RI)1IRUFROODJHQILEULOORJHQHVLVDQGUHFHQWSXEOLVKHGZRUNVKRZLQJWKDWFROODJHQ
RUJDQL]DWLRQFDQFRUUHODWHZLWKGLVHDVHRXWFRPHZHQH[WH[DPLQHGZKHWKHUWKH
FKDQJHVLQ(&0DFFXPXODWLRQREVHUYHGVKRUWWHUPin vitroWUDQVODWHGLQWRVLJQLILFDQW
FKDQJHVLQWKHVWUXFWXUHDQGDEXQGDQFHRIFROODJHQin vivo8VLQJ6HFRQG+DUPRQLF
*HQHUDWLRQ6+*ZHH[DPLQHGWKHVWUXFWXUHDQGDEXQGDQFHRIFROODJHQLQPXOWLSOH
WXPRUVWRLQYHVWLJDWHZKHWKHU0HQDDQG0HQD,19ZHUHDVVRFLDWHGZLWKFKDQJHVLQ
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
FROODJHQDEXQGDQFHDQGRUJDQL]DWLRQin vivo. :HH[DPLQHGWKHWKLFNQHVVRIWKH
FROODJHQFDSVXOHVXUURXQGLQJWKHWXPRUV,QWUDYLWDOLPDJLQJRIWXPRUVUHYHDOHGWKDW
GHOHWLRQRIWKH/(5(5LQERWK0HQDDQG0HQD,19WXPRUVOHDGWRVLJQLILFDQWO\
WKLFNHUFROODJHQFDSVXOHV)LJ6-6LPLODUO\NQRFNGRZQRI0HQDLQFHOOVDOVROHG
WRDQLQFUHDVHLQFROODJHQWKLFNQHVV)LJ6.+LVWRORJLFDODQDO\VLVRIWXPRUVHFWLRQV
VKRZHGWKDWWXPRUVLQ00793\07PLFHQXOOIRU0HQDNQRZQWRKDYHVLJQLILFDQWO\
ORZHUOHYHOVRIPHWDVWDVLVVKRZHGVLJQLILFDQWO\LQFUHDVHGFROODJHQOHYHOVFRPSDUHG
WRZLOGW\SHPLFH)LJ$%(QGRJHQRXV0HQD,19OHYHOVLQZLOGW\SH3\07WXPRUVZHUH
FRUUHODWHGZLWKORZHUOHYHOVRIFROODJHQ)LJ&',QSDWLHQWVZLWKKLJK0HQD,19
H[SUHVVLRQWKHUHZDVVLJQLILFDQWO\ORZHUFROODJHQVLJQDOWKDQLQSDWLHQWVZLWKORZ
0HQD,19H[SUHVVLRQ)LJ()7RJHWKHUWKHVHGDWDVXJJHVWWKDWKLJKO\LQYDVLYHWXPRUV
DULVLQJIURPRUFRUUHODWLQJZLWK0HQD,19H[SUHVVLRQZHUHDVVRFLDWHGZLWKWKLQQHU
FROODJHQFDSVXOHVZKLOHWKHSRRUO\PHWDVWDWLFWXPRUVDULVLQJIURP0HQDGHILFLHQWFHOOV
ZHUHDVVRFLDWHGZLWKWKLFNHUFROODJHQFDSVXOHV
:KLOHWKHDPRXQWRIFROODJHQGHSRVLWLRQVXUURXQGLQJWXPRUDQLPSRUWDQWIDFWRULQ
ORFDOLQYDVLRQFKDQJHVLQFROODJHQVKDSHDQGRULHQWDWLRQKDYHDOVREHHQOLQNHGZLWK
LQYDVLYHQHVV$ORZHUGHQVLW\RIFROODJHQRUJDQL]HGLQWRVWUDLJKWILEHUVRULHQWHG
SHUSHQGLFXODUO\WRWKHHGJHRIWKHWXPRUKDVEHHQDVVRFLDWHGZLWKSRRURXWFRPHLQD
EUHDVWFDQFHUFRKRUW5HSUHVHQWDWLYHLPDJHVRIWKHFROODJHQDWWKHHGJHRIWKH
0'$0%DQG680[HQRJUDIWWXPRUVVKRZ&RQWURODQG0HQDWXPRUV
KDGGHQVHFXUO\FROODJHQILEHUVRUJDQL]HGLQDOOGLUHFWLRQVUHODWLYHWRWKHWXPRUHGJH
)LJ*+)LJ6/+RZHYHU0HQD,19WXPRUVGLVSOD\HGDVLJQLILFDQWO\LQFUHDVHG
IUHTXHQF\RIILEHUVRULHQWDWHGSHUSHQGLFXODUWRWKHWXPRUHGJH)LJ*+
0HQDǻ/(5(5DQG0HQD,19ǻ/(5(5WXPRUVKDGFROODJHQILEHUVSDUDOOHOWRWKH
WXPRUHGJH)LJ6/0,QGHHGWKHVHWXPRUVFRQWDLQHGDEXQGDQWFROODJHQILEHUVZLWK
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
YHU\VPDOODQJOHVUHODWLYHWRWKHWXPRUHGJH%RWKSKHQRW\SHVZHUHDOVRREVHUYHGLQ
WXPRUVJHQHUDWHGZLWK680FHOOV)LJ*6127RJHWKHUWKHVHUHVXOWVLQGLFDWH
WKDWWKHLQLWLDO0HQD,19GHSHQGHQWFKDQJHVLQ(&0DFFXPXODWLRQREVHUYHGLQRXUVKRUW
WHUPin vitroDVVD\VFDQWUDQVODWHin vivoRYHUPXFKORQJHUWLPHVFDOHVDVVRFLDWHGZLWK
WXPRUSURJUHVVLRQLQWRVLJQLILFDQWUHGXFWLRQVLQRYHUDOODFFXPXODWLRQRIHQFDSVXODWLQJ
FROODJHQDFFRPSDQLHGE\LQFUHDVHGDEXQGDQFHRIOLQHDUILEHUVRULHQWHGSHUSHQGLFXODUO\
WRWKHWXPRUPDUJLQ
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
'LVFXVVLRQ
2XUUHVXOWVOHDGXVWRSURSRVHDQRYHOPHFKDQLVPE\ZKLFKWXPRUFHOOVVHQVHUHVSRQG
WRDQGUHRUJDQL]HWKH(&0WRVXSSRUWPHWDVWDVLVVXPPDUL]HGLQ)LJ,QWHJULQ
GHSHQGHQWRXWVLGHLQVLJQDOLQJDW);VDVZHOODVLQVLGHRXW(&0UHPRGHOLQJDUH
QHFHVVDU\IRUGLUHFWLRQDOPLJUDWLRQWRZDUGV)1)1DQGĮH[SUHVVLRQDUHXSUHJXODWHG
LQEUHDVWFDQFHUWXPRUVDQGKLJKOHYHOVRIWXPRU)1DQGĮKDYHEHHQDVVRFLDWHGZLWK
SRRURXWFRPHWKRXJKQRWE\WKHPVHOYHVLQWKHGDWDVHWVZHDQDO\]HG*LYHQWKH
KHWHURJHQHRXVOHYHOVRI)1ZLWKLQERWKWKHSULPDU\WXPRUDQGPHWDVWDWLFQLFKH
GLVVHPLQDWLQJFHOOVPXVWEHDEOHWRPLJUDWHHIIHFWLYHO\WKURXJKDUHDVRIGLIIHUHQW)1
FRQFHQWUDWLRQVDVZHOODVWRPRYHIURPDUHDVRIORZ)1WRDUHDVRIKLJK)1DQGYLFH
YHUVD,QDGGLWLRQWRVLPSO\EHLQJSHUPLVVLYHIRUPLJUDWLRQZHVKRZWKDW)1DFWVDVD
GLUHFWLRQDOFXHin vivo([SUHVVLRQRI0HQDGULYHVKDSWRWD[LVDWORZ)1FRQFHQWUDWLRQV
ZKLOHH[SUHVVLRQRI0HQD,19DOORZVFHOOVWRPLJUDWHDWKLJKFRQFHQWUDWLRQVRI)1VLPLODU
WRWKRVHSUHVHQWDURXQGEORRGYHVVHOVLQWXPRUV$EURJDWLRQRIWKHLQWHUDFWLRQ
EHWZHHQ0HQD0HQD,19DQGĮȕDEROLVKHGKDSWRWD[LVin vitro, ZKLOHDOVRVLJQLILFDQWO\
GHFUHDVLQJPHWDVWDVLVLQWZRPRXVH[HQRJUDIWEUHDVWFDQFHUPRGHOV7RJHWKHUWKHVH
ILQGLQJVVXSSRUWWKHLGHDWKDW)1FDQDFWDVDSRWHQWJXLGDQFHDQGPRWLOLW\FXHIRUWXPRU
FHOOVGXULQJPHWDVWDWLFSURJUHVVLRQ
$WDPHFKDQLVWLFOHYHOLQFOXVLRQRIWKH,19VHTXHQFHLQFUHDVHVWKHDVVRFLDWLRQ
RI0HQDZLWKĮE\IROG:HSURSRVHWKDWLQFUHDVHGDVVRFLDWLRQRI0HQD,19YV
0HQDZLWKĮOLNHO\XQGHUOLHVWKHLVRIRUPVSHFLILFGLIIHUHQFHVLQĮȕPHGLDWHGHIIHFWV
RQKDSWRWD[LVDQGIRFDOFRPSOH[HV7KH,19VHTXHQFHLVLQVHUWHGEHWZHHQWKHDPLQR
WHUPLQDO(9+GRPDLQLQ0HQDZKLFKPHGLDWHVLQWHUDFWLRQVZLWKVHYHUDORWKHU
PROHFXOHVDVVRFLDWHGZLWKLQWHJULQIXQFWLRQDQGWKH/(5(5GRPDLQZKLFKELQGVGLUHFWO\
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
WRĮ,WZLOOEHRIJUHDWLQWHUHVWWRGHWHUPLQHH[DFWO\KRZWKH,19VHTXHQFH
PRGLILHVLQWHUDFWLRQVZLWKĮ
7KH(&0FDQDOVRGHOLYHUVLJQDOVWRRWKHUW\SHVRIUHFHSWRUVE\SURYLGLQJELQGLQJ
VLWHVIRUWKHLUOLJDQGV:KLOHLWLVZHOOHVWDEOLVKHGWKDWSURWHRJO\FDQVGLUHFWO\ELQG
JURZWKIDFWRUVDOORZLQJWKH(&0WRDFWDVDUHVHUYRLUIRULQYDVLYHVLJQDOVHPHUJLQJ
HYLGHQFHVXJJHVWVWKDW(&0SURWHLQVFRQWDLQGRPDLQVWKDWPD\DOORZWKHPWRDFWLYDWH
UHFHSWRUW\URVLQHNLQDVHVGLUHFWO\)1PHGLDWHGDFWLYDWLRQRIĮȕFDQIRUH[DPSOHOHDG
WR+*)LQGHSHQGHQWDFWLYDWLRQRI0HWWRSURPRWHLQYDVLRQLQRYDULDQFDQFHUFHOOV,Q
DGGLWLRQH[SUHVVLRQRIPXWDQWSSURWHLQZKLFKLVPXWDWHGLQRIFDQFHUVFDQ
GULYHLQYDVLRQWKURXJKHQKDQFHG5&3GHSHQGHQWFRUHF\FOLQJRIĮȕZLWKVHYHUDO
UHFHSWRUW\URVLQHNLQDVHV57.VLQFOXGLQJ(*)5DQG0HW*LYHQWKDW0HQD,19
H[SUHVVLRQVHQVLWL]HVFHOOVWR(*)HQDEOLQJWKHPWRLQYDGHDQGPLJUDWHLQUHVSRQVHWR
ORZ(*)FRQFHQWUDWLRQVLWZLOOEHLQWHUHVWLQJWRLQYHVWLJDWHWKHSRWHQWLDOUROH
RIFURVVWDONZLWK57.VLJQDOLQJLQ0HQD,19GULYHQKDSWRWD[LV
7KHLPSRUWDQFHRIWXPRUFHOOGULYHQ)1ILEULOORJHQHVLVDQG(&0UHPRGHOLQJLQ
0HQD,19GULYHQKDSWRWD[LVLVVXUSULVLQJ)1SRO\PHUL]DWLRQLVUHTXLUHGIRUGHSRVLWLRQRI
FROODJHQ,DQGDQWLERG\ELQGLQJWRWKHFROODJHQELQGLQJVLWHRQ)1LQKLELWVFROODJHQ
ILEULOORJHQHVLV+HUHZHVKRZIRUWKHILUVWWLPHWKDWWXPRUFHOOPHGLDWHG)1
ILEULOORJHQHVLVLVUHTXLUHGIRUKDSWRWD[LVDSURFHVVGULYHQE\H[SUHVVLRQRI0HQD,19:H
DOVRVKRZWKHILUVWHYLGHQFHIRUWXPRUFHOOGULYHQFROODJHQUHRUJDQL]DWLRQLQYLYRDOVR
GULYHQE\0HQD,19DQGLWVLQWHUDFWLRQZLWKĮȕ7RJHWKHUWKHVHGDWDVXJJHVWWKDWWXPRU
FHOOVFDQSDYHWKHLURZQZD\WREORRGYHVVHOV:KLOHWKHPHFKDQLVPE\ZKLFK)1DQG
LQWHJULQVGULYHFROODJHQILEULOORJHQHVLVUHPDLQVLQFRPSOHWHO\XQGHUVWRRGRXUUHVXOWV
LQGLFDWHWKDW0HQD,19SURYLGHVDOLQNEHWZHHQLQWHJULQDFWLYDWLRQDQGWKHF\WRVNHOHWRQWR
GULYH(&0RUJDQL]DWLRQE\WXPRUFHOOV7KH0HQD,19GULYHQUHGXFWLRQLQFROODJHQ
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
HQFDSVXODWLRQDQGLQFUHDVHLQOLQHDUILEULOVUDGLDWLQJIURPWKHWXPRUSHULSKHU\LQWXUQ
IXUWKHUHQKDQFHPHWDVWDWLFSKHQRW\SHV2YHUDOORXUILQGLQJVKLJKOLJKWWKHSRWHQWLDO
LPSRUWDQFHRIELGLUHFWLRQDOLQWHJULQPHGLDWHGVLJQDOLQJLQWKHWXPRUFHOOFRPSDUWPHQWLQ
DGGLWLRQWRWKHSUHYLRXVO\FKDUDFWHUL]HGFRQWULEXWLRQVIURPVWURPDOFHOOVLQWKH
UHJXODWLRQRI(&0VWUXFWXUH
2XUGDWDDOVRVXSSRUWDUROHIRUWKHUHODWLRQVKLSEHWZHHQ0HQD,19ĮDQG)1LQ
KXPDQEUHDVWFDQFHU8VLQJDQLVRIRUPVSHFLILFDQWLERG\DQGELRLQIRUPDWLFDQDO\VLVRI
DYDLODEOH7&*$GDWDZHIRXQGWKDWKLJKH[SUHVVLRQOHYHOVRI0HQD,19DQG)1DUH
DVVRFLDWHGZLWKLQFUHDVHGUHFXUUHQFHDQGSRRURXWFRPHLQWZRKXPDQEUHDVWFDQFHU
FRKRUWV)XWXUHVWXGLHVRQODUJHUSDWLHQWFRKRUWVZLOOEHQHHGHGWRKHOSGHWHUPLQHWKH
XWLOLW\RI0HQD,19DVDGLDJQRVWLFDQGSURJQRVWLFPDUNHUDQGZKHWKHULWSURYLGHV
DGGLWLRQDOLQIRUPDWLRQZKHQXVHGLQFRQMXQFWLRQZLWKRWKHUWXPRUPDUNHUV$OWRJHWKHU
RXUILQGLQJVUHLQIRUFHWKHLPSRUWDQFHRI0HQD,19LQKXPDQEUHDVWFDQFHUDQGVXJJHVW
WKDWWDUJHWLQJWKHFKHPRWDFWLFDQGKDSWRWDWLFSDWKZD\VE\ZKLFK0HQD,19SURPRWHV
LQYDVLRQFRXOGEHXVHIXOWKHUDSHXWLFDOO\IRUPHWDVWDWLFEUHDVWFDQFHU
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
0DWHULDOVDQGPHWKRGV
$QWLERG\UHDJHQWVJURZWKIDFWRUVDQGLQKLELWRUV
$QWLERGLHVĮIRU,)%'%LRVFLHQFHVIRU,30LOOLSRUH$%DQGIRU:%
6DQWD&UX]%LRWHFKQRORJ\VFĮY%'%LRVFLHQFHVĮ$EFDP
DEĮ$EFDPDEȕ%'%LRVFLHQFHV)$.%'%LRVFLHQFHV
S)$.<,QYLWURJHQ*&OHDYHG&DVSDVH&67.L
&67)1%'%LRVFLHQFHVS&67FORQH&5&36LJPD6HHIRU
GHVFULSWLRQRI0HQD,19UDEELWPRQRFORQDODQWLERG\$QLPDOVZHUHLPPXQL]HGZLWKD
SHSWLGHFRQWDLQLQJWKHVHTXHQFHHQFRGHGE\WKH,19H[RQ&ORQHVZHUHVFUHHQHGIRU
0HQD,19VSHFLILFLW\LQ:HVWHUQEORWDVVD\VDQGE\LPPXQRVWDLQLQJRI))3(WXPRU
VHFWLRQVIURPZLOGW\SHRU0HQDQXOOPLFH)LJ6&LOHQJLWLGH6HOOHFN&KHPLFDOV
3'ĮEORFNLQJDQWLERG\'6+%)$.L6DQWD&UX]N'IUDJPHQWDQGLWVFRQWURO
SHSWLGHIRUEORFNDGHRIILEULOORJHQHVLVJLIWIURP'U6RWWLOH8QLYHUVLW\RI5RFKHVWHU)1
SXULILHGIURPDSODVPLGIURP52+
&HOOFXOWXUH
0'$0%FHOOVZHUHSXUFKDVHGGLUHFWO\IURP$7&&LQ-XQHZKHUHFHOOOLQHVDUH
DXWKHQWLFDWHGE\VKRUWWDQGHPUHSHDWSURILOLQJ7KHVHFHOOVQRWUHDXWKHQWLFDWHGE\RXU
ODEDQGZHUHFXOWXUHGLQ'0(0ZLWK)%6+\FORQH680FHOOVZHUHREWDLQHG
IURP-RDQ%UXJJH¶VODEDW+DUYDUG0HGLFDO6FKRRO-DQXDU\DQGZHUHQRWUH
DXWKHQWLFDWHGLQRXUODE680FHOOVZHUHFXOWXUHGDFFRUGLQJWRWKH$7&&SURWRFROV
09'ILEUREODVWVFHOOVZHUHLVRODWHGIURPPLFHLQRXUODELQ2FWREHUDQGFXOWXUHGDV
SUHYLRXVO\GHVFULEHG7KHVHFHOOOLQHVZHUHDXWKHQWLFDWHGLQRXUODEDQGGHOHWLRQRI
0HQDZDVYHULILHGDWWKHP51$DQGSURWHLQOHYHO5HWURYLUDOSDFNDJLQJLQIHFWLRQDQG
)$&6ZHUHSHUIRUPHGDVSUHYLRXVO\GHVFULEHG&HOOOLQHVZHUHHQJLQHHUHGWR
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
H[SUHVV0HQDLVRIRUPVVWDEO\DWIROGKLJKHUOHYHOVWKDQSDUHQWDOOLQHV09'
ILEUREODVWVZHUHLVRODWHGZHUHPDLQWDLQHGDWƒ&&2LQ'0(VXSSOHPHQWHGZLWK
O*OXWDPLQHSHQLFLOOLQDQGVWUHSWRP\FLQIHWDOERYLQHVHUXPDQG8POLQWHUIHURQ
,6LJPD6WDEOH.QRFNGRZQFHOOOLQHVZHUHJHQHUDWHGXVLQJPLUEDVHG
VK51$VHTXHQFHµ&$*$$*$&$$7&*&&&777$$¶IRU0HQDH[SUHVVLQJDQP&KHUU\
WDJ
,PPXQRKLVWRFKHPLVWU\
)L[DWLRQSURFHVVLQJDQGVWDLQLQJRIWLVVXHVHFWLRQVIURPWXPRUVZDVFDUULHGRXWDV
SUHYLRXVO\GHVFULEHG7XPRUVGLVVHFWHGIURP12'6&,'PLFHZHUHIL[HGLQ
EXIIHUHGIRUPDOLQDQGHPEHGGHGLQSDUDIILQ7LVVXHVHFWLRQVȝPWKLFNZHUH
GHSDUDIILQL]HGIROORZHGE\DQWLJHQUHWULHYDOXVLQJ&LWUD3OXVVROXWLRQ%LRJHQH[$IWHU
HQGRJHQRXVSHUR[LGDVHLQDFWLYDWLRQVHFWLRQVZHUHLQFXEDWHGZLWKSULPDU\DQWLERGLHV
RYHUQLJKWDWƒ&DQGIOXRUHVFHQWO\ODEHOHGVHFRQGDU\DQWLERGLHVDWURRPWHPSHUDWXUHIRU
KUV6HFWLRQVZHUHVWDLQHGXVLQJWKHIROORZLQJDQWLERGLHVDQWL0HQDDQWL.L
%'%LRVFLHQFHVFOHDYHG&DVSDVH%'%LRVFLHQFHV)OXRURFKURPHVRQVHFRQGDU\
DQWLERGLHVLQFOXGHG$OH[D)OXRU$OH[D)OXRUDQG$OH[D)OXRU-DFNVRQ
,PPXQRUHVHDUFK6HFWLRQVZHUHPRXQWHGLQ)OXRURPRXQWPRXQWLQJPHGLDDQGLPDJHG
DWURRPWHPSHUDWXUH=VHULHVRILPDJHVZHUHWDNHQRQDQ$SSOLHGSUHFLVLRQ'HOWD9LVLRQ
PLFURVFRSHXVLQJ6RIWZRU[DFTXLVLWLRQDQ2O\PSXV[1$SODQDSRREMHFWLYHDQGD
3KRWRPHWULFV&RRO61$3+4FDPHUD,PDJHVZHUHGHFRQYROYHGXVLQJ'HOWDYLVLRQ
6RIWZRU[VRIWZDUHDQGREMHFWLYHVSHFLILFSRLQWVSUHDGIXQFWLRQ$WOHDVWLPDJHVZHUH
FDSWXUHGIRUHDFKWXPRUZLWKDWOHDVWWXPRUVSHUWXPRUJURXS
70$
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
'HWDLOVRIWKHSDWLHQWFRKRUWDQGDVVRFLDWHGGDWDXVHGWRJHQHUDWHWKH70$DUH
SXEOLVKHG7KH70$ZDVVWDLQHGE\LPPXQRIOXRUHVFHQFHDQGLPDJHGZLWKD
9HFWUDDXWRPDWHGVOLGHVFDQQHUDQGD;REMHFWLYH7KHILHOGRIYLHZZLWKWKLV
REMHFWLYHFRYHUVRIWKHFRUHVSRW(DFKSDWLHQWKDGWKUHHFRUHVRQWKH70$
$OOZHUHLPDJHGEXWVRPHKDGWRUHPRYHGGXHWRODFNRIWLVVXHRUIROGHGWLVVXH
)OXRUHVFHQFHLQWHQVLW\LQWKHWXPRUFRPSDUWPHQWZDVDQDO\]HGXVLQJ,QIRUPVRIWZDUH
0HQD,19DQG)1LQWHQVLW\PHWULFVDUHLQDUELWUDU\XQLWV
+DSWRWD[LVDVVD\V
0LFURIOXLGLFGHYLFHVZHUHSUHSDUHGDVGHVFULEHG)RUKDSWRWD[LVRQD'PDWUL[
DIWHUERQGLQJ3'06GHYLFHVWR0DWWHNGLVKHVWKHFKDPEHUZDVFRDWHGZLWKPJPO
&ROODJHQ,IRUKUDWƒ&DQGWKHQȝJPOIOXRUHVFHQWO\ODEHOHG)1ZDVIORZHG
WKURXJKWKHVRXUFHFKDQQHOIRUKU&HOOVZHUHWKHQSODWHGLQWKHGHYLFHLQIXOOVHUXP
PHGLDDQGOHIWWRDWWDFKIRUKUEHIRUHLPDJLQJ)RUKDSWRWD[LVLQD'PDWUL[FHOOVZHUH
UHVXVSHQGHGLQPJPOFROODJHQ,%'%LRVFLHQFHVZLWK;'0(0DQG11D2+DQG
Q0(*)LQIXOOVHUXPPHGLDSODWHGLQWKHFHOOFXOWXUHFKDPEHUDQGOHIWWRVHWWOHIRU
KUVDWƒ&)OXRUHVFHQWO\ODEHOHG)1ZDVWKHQIORZHGWKURXJKWKHVRXUFHFKDQQHOIRU
KUEHIRUHLPDJLQJ)RUDOOKDSWRWD[LVH[SHULPHQWVWKH)1FRQFHQWUDWLRQUHSUHVHQWVWKH
FRQFHQWUDWLRQDWWKHWRSRIWKHJUDGLHQW8QOHVVPHQWLRQHG)1FRQFHQWUDWLRQDWWKHWRS
RIWKHJUDGLHQWLVȝJPO&HOOVZHUHLPDJHGRYHUQLJKWLQWKHKDSWRWD[LVGHYLFHZLWK
LPDJHVEHLQJDFTXLUHGHYHU\PLQIRUKULQDQHQYLURQPHQWDOO\FRQWUROOHGPLFURVFRSH
7(1LNRQZLWKD;REMHFWLYHDQGD3KRWRPHWULFV&RROVQDS+4FDPHUD
,QGLYLGXDOFHOOVZHUHPDQXDOO\WUDFNHGXVLQJ,PDJH-VRIWZDUH0DQXDO7UDFNLQJSOXJLQ
7KHWUDFNVREWDLQHGZHUHDQDO\]HGXVLQJWKH&KHPRWD[LV7RRO,PDJH-SOXJLQIURP
,ELGL7KLVDQDO\VLVWRROZDVXVHGWRH[WUDFWWKH)0,)LJ$DORQJZLWKWKHYHORFLW\RI
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
PLJUDWLRQDQGWKHSHUVLVWHQFHRIPLJUDWLRQXVLQJWKH'7UDWLRQHWSDWKOHQJWKWRWDOSDWK
OHQJWK
7XPRUIRUPDWLRQDQGPHWDVWDVLVDVVD\
$OODQLPDOH[SHULPHQWVZHUHDSSURYHGE\WKH0,7'LYLVLRQRI&RPSDUDWLYH0HGLFLQH)RU
[HQRJUDIWH[SHULPHQWV0'$0%RU680FHOOVPLOOLRQSHUPRXVHLQ3%6DQG
FROODJHQ,H[SUHVVLQJGLIIHUHQW0HQDLVRIRUPVZHUHLQMHFWHGLQWRWKHWKULJKW
PDPPDU\IDWSDGRIVL[ZHHNROGIHPDOH12'6&,'PLFH7DFRQLF7XPRUVL]HZDV
PHDVXUHGZHHNO\ZLWKFDOLSHUVRUZHHNVSRVWVXUJHU\RQFHWXPRUVKDGUHDFKHG
FPLQGLDPHWHUPLFHZHUHXVHGIRULQWUDYLWDOLPDJLQJDQGWKHQVDFULILFHGDQGWKHLU
WXPRUVDQGOXQJVZHUHIL[HGLQIRUPDOLQRYHUQLJKW0HWDVWDWLFLQGH[ZDVFDOFXODWHG
E\FRXQWLQJWKHQXPEHURIPHWDVWDVHVLQHDFKOREHUHODWLYHWRWKHZHLJKWRIWKHWXPRU
(DFKWXPRUJURXSFRQWDLQHGPLFH3\070079PLFHZHUHREWDLQHGIURP-DFNVRQ
PLFHZHUHOHIWWRJURZWKIRUZHHNVZKHQWKH\KDGGHYHORSHGWXPRUVRIDERXW
FPLQGLDPHWHU+LVWRORJLFDODQDO\VLVRI+(VIURPWKHVHWXPRUVFRQILUPHGWKHVHWXPRUV
ZHUHDGYDQFHGFDUFLQRPD
In vivoLQYDVLRQDVVD\
7KHin vivoLQYDVLRQDVVD\ZDVSHUIRUPHGLQDWOHDVWPLFHSHUFRQGLWLRQDVSUHYLRXVO\
GHVFULEHG %ULHIO\ QHHGOHV ZHUH KHOG LQ SODFH E\ D PLFURPDQLSXODWRU DURXQG D
VLQJOH PDPPDU\ WXPRU RI DQ DQHVWKHWL]HG PRXVH 1HHGOHV FRQWDLQHG D PL[WXUH RI
PJPO &ROODJHQ , ('7$ ZLWK / PHGLD RU LQFUHDVLQJDPRXQWV ZLWK )1 $IWHU IRXU
KRXUV WKH FRQWHQWV RI WKH QHHGOHV ZHUH H[WUXGHG &HOOV ZHUH VWDLQHG ZLWK '$3, DQG
FRXQWHG
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
,QWUDYLWDO,PDJLQJDQGin vivoKDSWRWD[LV
,QWUDYLWDOPXOWLSKRWRQLPDJLQJZDVSHUIRUPHGDVGHVFULEHGSUHYLRXVO\XVLQJD[
1$ZDWHULPPHUVLRQREMHFWLYHZLWKFRUUHFWLRQOHQV)RUin vivoKDSWRWD[LVD
PLFURVFDOHGHYLFHILOOHGZLWKPXOWLSOHUHVHUYRLUVZLWKSRZGHUHGUKRGDPLQH)1RU
GH[WUDQZDVSUHSDUHG$IWHUH[SRVLQJWKHWXPRUWKHGHYLFHZDVLPSODQWHGLQWRWKH
WXPRUHGJH+RXUORQJWLPHODSVHPRYLHVZHUHDQDO\]HGIRUIUHTXHQF\RIPRWLOLW\DQG
WUDFNLQJDQGWRPHDVXUHDQGTXDQWLI\FHOOFKDUDFWHULVWLFVLQ'DQGRYHUWLPHXVLQJ1,+
,PDJH-&HOOVWKDWZHUHHLWKHUSURWUXGLQJRUPRYLQJZHUHFRXQWHGDVPRWLOH)RUHDFK
PRYLHWKH)0,ZDVFDOFXODWHGZLWKWKHDQJOHIRUHDFKWUDFNPDGHUHODWLYHWRWKH
GLUHFWLRQRI)1JUDGLHQWIRUHDFKIUDPH'DWDLVSRROHGIURPPLFHSHUWXPRUJURXS
ZLWKILHOGVLPDJHGSHUPRXVHZLWKDWRWDORIDWOHDVWFHOOVSHUWXPRUW\SH
WUDFNHG&ROODJHQVLJQDOZDVYLVXDOL]HGE\VHFRQGKDUPRQLFJHQHUDWLRQDQGLPDJHV
ZHUHDQDO\]HGXVLQJ&7)LUHVRIWZDUHWRFDOFXODWHFROODJHQILEHURULHQWDWLRQZLGWK
DQGOHQJWK'DWDZHUHSRROHGIURPPLFHSHUWXPRUJURXSZLWKILHOGVLPDJHG
SHUPRXVH
:HVWHUQ%ORW,PPXQRSUHFLSLWDWLRQ
)RUWKHĮLPPXQRSUHFLSLWDWLRQ0HQDDQG0HQD,19ZHUHO\VHGZLWK&6.EXIIHU
P0 3,3(6 ± S+ P0 1D&O P0 VXFURVH P0 0J&O P0 0Q&O
7ULWRQ;SURWHDVHDQGSKRVSKDWDVHLQKLELWRUVDQGSDVVHGWKURXJKDJDXJH
QHHGOH /\VDWHV ZHUH SUHFOHDUHG ZLWK SURWHLQ $ EHDGV IRU K LQFXEDWHG ZLWK WKH
LQWHJULQĮDQWLERG\0LOOLSRUHIRUKDWƒ&DQGWKHQFDSWXUHGZLWK%6$
EORFNHG SURWHLQ $ EHDGV IRU K %HDGV ZHUH ZDVKHG WKUHH WLPHV LQ &6. O\VLV EXIIHU
DQGSURWHLQVZHUHHOXWHGLQ[VDPSOHEXIIHU6WDQGDUGSURFHGXUHVZHUHXVHGIRUSURWHLQ
HOHFWURSKRUHVLV ZHVWHUQ EORWWLQJ DQG LPPXQRSUHFLSLWDWLRQ 0'$0% H[SUHVVLQJ
GLIIHUHQW 0HQD LVRIRUPV ZHUH O\VHG LQ P0 7ULV P0 1D&O JO\FHURO 13
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
DQG 0 ('7$ ZLWK D SURWHDVH 0LQLFRPSOHWH SURWHDVH LQKLELWRUV 5RFKH DQG D
SKRVSKDWDVH LQKLELWRU FRFNWDLO 3KRV6723 5RFKH DW ƒ& 3URWHLQ O\VDWHV ZHUH
VHSDUDWHG E\ 6'63$*( WUDQVIHUUHG WR D QLWURFHOOXORVH PHPEUDQH EORFNHG ZLWK
2G\VVH\ %ORFNLQJ %XIIHU /L&RU LQFXEDWHG LQ SULPDU\ DQWLERG\ RYHUQLJKW DW ƒ&
3URWHLQV ZHUH GHWHFWHG XVLQJ /LFRU VHFRQGDU\ DQWLERGLHV 3URWHLQ OHYHO LQWHQVLW\ ZDV
PHDVXUHGZLWK,PDJH-DQGGDWDZHUHSRROHGIURPDWOHDVWGLIIHUHQWH[SHULPHQWV
)$&V
0'$0%FHOOVH[SUHVVLQJWKHGLIIHUHQWLVRIRUPVZHUHWU\SVLQL]HGUHVXVSHQGHGLQ
PHGLDDQGWKHQLQFXEDWHGZLWKDSULPDU\DQWLERG\LQ3%6DQGPHGLDIRUPLQVRQ
LFH1H[WWKHFHOOVZHUHLQFXEDWHGZLWKDVSHFLHVDSSURSULDWH$OH[DWDJJHG
VHFRQGDU\DQWLERG\DQGWKHQUHVXVSHQGHGLQ3%6ZLWKȝJPOSURSLGLXPLRGLGH
6DPSOHVZHUHWKHQDQDO\]HGRQD)$&6&DOLEXUPDFKLQH%'%LRVFLHQFHV'DWDLV
SRROHGIURPDWOHDVWVHSDUDWHH[SHULPHQWVZLWKFHOOVDQDO\]HGSHUH[SHULPHQW
,PPXQRIOXRUHVFHQFH
&HOOVZHUHSODWHGLQDKDSWRWD[LVGHYLFHRQDȝJPO')1JUDGLHQWIRUKURURQ
FROODJHQFRDWHGJODVVERWWRPHGGLVKHV0DW7HNLQVHUXPIUHHPHGLDIRUPLQDW
ƒ&&HOOVZHUHWKHQIL[HGIRUPLQLQSDUDIRUPDOGHK\GHLQ3+(0EXIIHUWKHQ
SHUPHDELOL]HGZLWK7ULWRQ;EORFNHGZLWK%6$DQGLQFXEDWHGZLWKSULPDU\
DQWLERGLHVRYHUQLJKWDWƒ&=VHULHVRILPDJHVZHUHWDNHQRQDQ$SSOLHG3UHFLVLRQ
'HOWD9LVLRQPLFURVFRSHXVLQJ6RIWZRU[DFTXLVLWLRQDQ2O\PSXV[1$SODQDSR
REMHFWLYHDQGD3KRWRPHWULFV&RRO61$3+4FDPHUD,PDJHVZHUHGHFRQYROYHGXVLQJ
'HOWDYLVLRQ6RIWZRU[VRIWZDUHDQGREMHFWLYHVSHFLILFSRLQWVSUHDGIXQFWLRQ,PDJHVZHUH
DQDO\]HGZLWK,PDJH-,PDJHVDUHSRROHGIURPDWOHDVWLQGHSHQGHQWH[SHULPHQWVDW
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
OHDVWFHOOVSHUH[SHULPHQW
0HQD,197&*$GDWDUHWULHYDO
51$VHTGDWDLQIDVWTIRUPDWZHUHREWDLQHGIURP7&*$)RUHDFKVDPSOH(1$+
0HQDGHULYHGUHDGVZHUHH[WUDFWHGIURPWKHIXOOGDWDVHWE\DOLJQLQJWRDWDUJHW
GDWDEDVHWKDWFRQWDLQHGFROOHFWLRQRIDOOSRVVLEOH(1$+LVRIRUPVXVLQJ%:$YHUVLRQ
3URSHUO\SDLUHG(1$+UHDGVZHUHWKHQH[WUDFWHGZLWK6DPWRROVYHUVLRQ
(1$+LVRIRUPVZHUHWKHQTXDQWLILHGE\DOLJQLQJWRKJXVLQJWRSKDWYHUVLRQ
JXLGHGZLWKDQHGLWHG*7)ILOHGHULYHGIURPWKH86&6NQRZQJHQHVDQQRWDWLRQWKDW
FRQWDLQHGDOO(1$+YDULDQWVRILQWHUHVW%HGWRROVYHUVLRQDQGDFXVWRPS\WKRQ
VFULSWZHUHWKHQXVHGWRFRXQWUHDGVWKDWRYHUODSZLWKHDFK(1$+H[RQ7KHUHVXOWLQJ
FRXQWVSHUH[RQZHUHWKHQQRUPDOL]HGIRU51$ORDGLQJE\FDOFXODWLQJDFRXQWVSHU
PLOOLRQUHDGVSHU.ERIP51$XVLQJDVXPRIH[RQOHYHOFRXQWVLQWKHSXEOLFO\DYDLODEOH
DQGSUHSURFHVVHG7&*$GDWDDVWKHWRWDODOLJQHGFRXQWVGHQRPLQDWRU
6XUYLYDOUHFXUUHQFHGDWDDQDO\VLV
7KHUHODWLRQVKLSEHWZHHQ0HQD0HQD,19H[SUHVVLRQOHYHOVIURPP51$7&*$RUSURWHLQ
70$DQGVXUYLYDOWLPHWRGHDWKRUPHWDVWDVLVWLPHWRUHFXUUHQFHZDVDVVHVVHGE\
/RJUDQN0DQWHO&R[WHVW,QHDFKVDPSOHVSDWLHQWVZHUHELQQHGLQWRTXDUWLOHV
DFFRUGLQJ0HQDRU0HQD,19H[SUHVVLRQ4EHLQJWKHKLJKHVWOHYHORIH[SUHVVLRQDQG4
EHLQJWKHORZHVW7KHKD]DUGUDWLRIRUHDFKTXDUWLOHZLWKFRQILGHQFHLQWHUYDO
YDOXHVZDVFDOFXODWHG7KHSYDOXHJHQHUDWHGE\WKLVORJUDQNWHVWHYDOXDWHVZKHWKHU
WKHGLIIHUHQFHLQWKHFXUYHVLVVLJQLILFDQWO\GLIIHUHQW:HDOVRSHUIRUPHGWKH/RJUDQN
WHVWIRUWUHQGWRIXUWKHUDVVHVVWKHGLIIHUHQFHVEHWZHHQWKHFXUYHVUHSUHVHQWLQJSDWLHQWV
ZLWKYDU\LQJOHYHOVRI0HQDLVRIRUPH[SUHVVLRQ
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
7KHKD]DUGHIIHFWVRI0HQD,19DQG0HQDXSRQWKHWLPHWRGHDWKZHUH
LQYHVWLJDWHGE\&R[UHJUHVVLRQXVLQJ5EDVLQJRQ7&*$%5&$GDWD,QRUGHUWR
PDNHFRPSDULVRQDFURVVYDULDEOHVZHILUVWVWDQGDUGL]HG0HQDDQG0HQD,1953.0
YDOXHVWRPHDQ]HURDQGVWDQGDUGGHYLDWLRQRQH&R[UHJUHVVLRQVZHUHWKHQFDUULHGRXW
EDVLQJRQWKHVWDQGDUGL]HG0HQD,19YDOXHVRUWKHVWDQGDUGL]HG0HQD53.0YDOXHVDV
WKHRQO\LQGHSHQGHQWYDULDEOHWRSUHGLFWWKHHIIHFWVXSRQWKHWLPHRIGHDWKRIWKH%5&$
SDWLHQWVLQ7&*$VWXG\7KHDVVRFLDWLRQEHWZHHQ0HQD,190HQDH[SUHVVLRQOHYHODQG
VXUYLYDOVWDWXVRI7&*$%5&$VXEMHFWVZDVHYDOXDWHGE\ORJLVWLFUHJUHVVLRQXVLQJ5
,QRUGHUWRFRPSDUHFRHIILFLHQWVDFURVVWHVWVZHILUVWVWDQGDUGL]HG,19DQG
0HQDYDOXHVWREHPHDQ]HURDQGVWDQGDUGGHYLDWLRQRQH/RJLVWLFUHJUHVVLRQVZHUH
FRQGXFWHGE\FKRRVLQJVXUYLYDOVWDWXVDVGHSHQGHQWYDULDEOHDVGHDWKDQGDV
DOLYH7KHRQO\LQGHSHQGHQWYDULDEOHILWWHGLQWKHPRGHOZDV,19RU0HQD
UHVSHFWLYHO\3YDOXHVDQGFRHIILFLHQWVFRUUHVSRQGLQJWRWKHLQGHSHQGHQWYDULDEOHVZHUH
XVHGWRMXGJHWKHVLJQLILFDQFHRIWKHDVVRFLDWLRQDVZHOODVWKHVWUHQJWKRIWKH
DVVRFLDWLRQ
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
$XWKRUFRQWULEXWLRQV
0-2GHVLJQHGDQGSHUIRUPHGH[SHULPHQWVGDWDDQDO\VLVDQGSUHSDUHGWKHPDQXVFULSW
)%*JXLGHGRYHUDOOH[SHULPHQWDOGHVLJQSHUIRUPHG7&*$GDWDDQDO\VLVDQGKHOSHG
SUHSDUHWKHPDQXVFULSW2-GHYHORSHGWKHPLFURVFDOHGHYLFHDQGKHOSHGGHVLJQin vivo
KDSWRWD[LVH[SHULPHQWV7.DQG/&%SHUIRUPHGKDSWRWD[LVDVVD\V
LPPXQRIOXRUHVFHQFHZHVWHUQEORWV%&*SHUIRUPHGWKH0HQDĮ,3H[SHULPHQWV-:
DVVLVWHGZLWKLQWUDYLWDOLPDJLQJDQGLQWUDYLWDOLPDJLQJ'5GHYHORSHGDQGYDOLGDWHGWKH
VK0HQDFRQVWUXFWV-0/KHOSHGZLWKDQLPDOH[SHULPHQWV6%$KHOSHGZLWKin vitro
KDSWRWD[LVDVVD\V&:DQG'0SHUIRUPHG7&*$GDWDUHWULHYDODQGDQDO\VLV.%:
JHQHUDWHGWKH70$5/0-&-(%'$/3.52+DQG)%*ZHUHLQYROYHGLQWKHVWXG\
GHVLJQDQGGDWDLQWHUSUHWDWLRQ$OODXWKRUVGLVFXVVHGWKHUHVXOWVDQGFRPPHQWHGRQWKH
PDQXVFULSW
$FNQRZOHGJHPHQWV
:HWKDQNWKH0LFURVFRS\)ORZ&\WRPHWU\DQG+LVWRORJ\IDFLOLWLHVLQWKH.,6ZDQVRQ
%LRWHFKQRORJ\&HQWHUIRUVXSSRUW:HWKDQN0HWD6WDWIRUXVHRID9HFWUDVFDQQHUDQG
VRIWZDUH:HWKDQN-HQQ\7DGURVIRUKHOSZLWKFHOOOLQHJHQHUDWLRQ6KDQQRQ+XJKHVIRU
WKHFORQLQJRI0HQDVK51$FRQVWUXFWVDQG-DQH6RWWLOH5RFKHVWHUIRUWKHN')1
IUDJPHQW
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
5HIHUHQFHV
-R\FH-$3ROODUG-:0LFURHQYLURQPHQWDOUHJXODWLRQRIPHWDVWDVLV1DW5HY
&DQFHU±
3LFNXS0:0RXZ-.:HDYHU907KHH[WUDFHOOXODUPDWUL[PRGXODWHVWKH
KDOOPDUNVRIFDQFHU(0%25HS(0%23UHVV±
1DED$&ODXVHU.5/DPDU-0&DUU6$+\QHV52([WUDFHOOXODUPDWUL[
VLJQDWXUHVRIKXPDQPDPPDU\FDUFLQRPDLGHQWLI\QRYHOPHWDVWDVLVSURPRWHUV
(OLIHH
&RQNOLQ0:.HHO\3-:K\WKHVWURPDPDWWHUVLQEUHDVWFDQFHULQVLJKWVLQWR
EUHDVWFDQFHUSDWLHQWRXWFRPHVWKURXJKWKHH[DPLQDWLRQRIVWURPDOELRPDUNHUV
&HOO$GK0LJU±
&RQNOLQ0:(LFNKRII-&5LFKLQJ.03HKONH&$(OLFHLUL.:3URYHQ]DQR33HW
DO$OLJQHGFROODJHQLVDSURJQRVWLFVLJQDWXUHIRUVXUYLYDOLQKXPDQEUHDVW
FDUFLQRPD$P-3DWKRO±
,RDFKLP(&KDUFKDQWL$%ULDVRXOLV(.DUDYDVLOLV97VDQRX+$UYDQLWLV'/HW
DO,PPXQRKLVWRFKHPLFDOH[SUHVVLRQRIH[WUDFHOOXODUPDWUL[FRPSRQHQWVWHQDVFLQ
ILEURQHFWLQFROODJHQW\SH,9DQGODPLQLQLQEUHDVWFDQFHU7KHLUSURJQRVWLFYDOXH
DQGUROHLQWXPRXULQYDVLRQDQGSURJUHVVLRQ(XU-&DQFHU±
%DH<..LP$.LP0.&KRL-(.DQJ6+/HH6-)LEURQHFWLQH[SUHVVLRQLQ
FDUFLQRPDFHOOVFRUUHODWHVZLWKWXPRUDJJUHVVLYHQHVVDQGSRRUFOLQLFDORXWFRPHLQ
SDWLHQWVZLWKLQYDVLYHEUHDVWFDQFHU+XP3DWKRO±
5RXVVRV(7&RQGHHOLV-63DWVLDORX$&KHPRWD[LVLQFDQFHU1DW5HY&DQFHU
±
$VWURI6&URZOH\'*HRUJH(/)XNXGD76HNLJXFKL.+DQDKDQ'HWDO'LUHFW
WHVWRISRWHQWLDOUROHVRI(,,,$DQG(,,,%DOWHUQDWLYHO\VSOLFHGVHJPHQWVRI
ILEURQHFWLQLQSK\VLRORJLFDODQGWXPRUDQJLRJHQHVLV0RO&HOO%LRO±
/HYHQWDO.5<X+.DVV//DNLQV-1(JHEODG0(UOHU-7HWDO0DWUL[
&URVVOLQNLQJ)RUFHV7XPRU3URJUHVVLRQE\(QKDQFLQJ,QWHJULQ6LJQDOLQJ&HOO
±
+XWWHQORFKHU$+RUZLW]$5,QWHJULQVLQFHOOPLJUDWLRQ&ROG6SULQJ+DUE3HUVSHFW
%LRO±
6FKZDU]EDXHU-('H6LPRQH':)LEURQHFWLQVWKHLUILEULOORJHQHVLVDQGLQYLYR
IXQFWLRQV&ROG6SULQJ+DUE3HUVSHFW%LRO±
.DGOHU.(+LOO$&DQW\/DLUG(*&ROODJHQILEULOORJHQHVLVILEURQHFWLQLQWHJULQV
DQGPLQRUFROODJHQVDVRUJDQL]HUVDQGQXFOHDWRUV&XUU2SLQ&HOO%LRO
±
*HUWOHU)&RQGHHOLV-0HWDVWDVLVWXPRUFHOOVEHFRPLQJ0(1$FLQJ7UHQGV&HOO
%LRO±
*XSWRQ6/5LTXHOPH'+XJKHV$OIRUG6.7DGURV-5XGLQD66+\QHV52HW
DO0HQDELQGVĮLQWHJULQGLUHFWO\DQGPRGXODWHVĮȕIXQFWLRQ-&HOO%LRO
±
*RVZDPL63KLOLSSDU86XQ'3DWVLDORX$$YUDKDP-:DQJ:HWDO
,GHQWLILFDWLRQRILQYDVLRQVSHFLILFVSOLFHYDULDQWVRIWKHF\WRVNHOHWDOSURWHLQ0HQD
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
SUHVHQWLQPDPPDU\WXPRUFHOOVGXULQJLQYDVLRQLQYLYR&OLQ([S0HWDVWDVLV
±
3KLOLSSDU85RXVVRV(72VHU0<DPDJXFKL+.LP+'*LDPSLHUL6HWDO$
0HQD,QYDVLRQ,VRIRUP3RWHQWLDWHV(*),QGXFHG&DUFLQRPD&HOO,QYDVLRQDQG
0HWDVWDVLV'HY&HOO±
5RXVVRV(7%DOVDPR0$OIRUG6.:\FNRII-%*OLJRULMHYLF%:DQJ<HWDO
0HQDLQYDVLYH0HQD,19SURPRWHVPXOWLFHOOXODUVWUHDPLQJPRWLOLW\DQG
WUDQVHQGRWKHOLDOPLJUDWLRQLQDPRXVHPRGHORIEUHDVWFDQFHU-&HOO6FL
±
:X&$VRNDQ6%%HUJLQVNL0(+D\QHV(06KDUSOHVV1(*ULIILWK-'HWDO
$USLVFULWLFDOIRUODPHOOLSRGLDDQGUHVSRQVHWRH[WUDFHOOXODUPDWUL[FXHVEXWLV
GLVSHQVDEOHIRUFKHPRWD[LV&HOO±
$VRNDQ6%-RKQVRQ+(5DKPDQ$.LQJ6-5RWW\-'/HEHGHYD,3HWDO
0HVHQFK\PDO&KHPRWD[LV5HTXLUHV6HOHFWLYH,QDFWLYDWLRQRI0\RVLQ,,DWWKH
/HDGLQJ(GJHYLDD1RQFDQRQLFDO3/&Ȗ3.&Į3DWKZD\'HY&HOO±
%HDU-(/RXUHLUR--/LERYD,,)lVVOHU55:HKODQG--*HUWOHU)%)%1HJDWLYH
UHJXODWLRQRIILEUREODVWPRWLOLW\E\(QD9$63SURWHLQV&HOO±
/H\PH$0DULYLQ$3HUH]*XWLHUUH]/1JX\HQ/7*DUFLD0DUFRV0,QWHJULQV
DFWLYDWHWULPHULF*SURWHLQVYLDWKHQRQUHFHSWRUSURWHLQ*,9*LUGLQ-&HOO%LRO
±
:DQJ:*RVZDPL6/DSLGXV.:HOOV$/:\FNRII-%6DKDL(HWDO
,GHQWLILFDWLRQDQGWHVWLQJRIDJHQHH[SUHVVLRQVLJQDWXUHRILQYDVLYHFDUFLQRPD
FHOOVZLWKLQSULPDU\PDPPDU\WXPRUV&DQFHU5HV±
+\QHV52<DPDGD.0)LEURQHFWLQV0XOWLIXQFWLRQDOPRGXODUJO\FRSURWHLQV-
&HOO%LRO±
0XOOHU3$-&DVZHOO37'R\OH%,ZDQLFNL037DQ(+.DULP6HWDO0XWDQW
S'ULYHV,QYDVLRQE\3URPRWLQJ,QWHJULQ5HF\FOLQJ&HOO±
2XGLQ0-+XJKHV6.5RKDQL10RXIDUUHM01-RQHV-*&RQGHHOLV-6HWDO
&KDUDFWHUL]DWLRQRIWKHH[SUHVVLRQRIWKHSURPHWDVWDWLF0HQD,19LVRIRUPGXULQJ
EUHDVWWXPRUSURJUHVVLRQ&OLQ([S0HWDVWDVLV'HF(SXEDKHDGRISULQW
:\FNRII-%6HJDOO-(&RQGHHOLV-67KHFROOHFWLRQRIWKHPRWLOHSRSXODWLRQRI
FHOOVIURPDOLYLQJWXPRU&DQFHU5HV
-RQDV2/DQGU\+0)XOOHU-(6DQWLQL-7%DVHOJD-7HSSHU5,HWDO$Q
LPSODQWDEOHPLFURGHYLFHWRSHUIRUPKLJKWKURXJKSXWLQYLYRGUXJVHQVLWLYLW\WHVWLQJ
LQWXPRUV6FL7UDQVO0HGUD
3LJQDWHOOL-*RVZDPL6-RQHV-*5RKDQ7(3LHUL(&KHQ;HWDO,QYDVLYH
EUHDVWFDUFLQRPDFHOOVIURPSDWLHQWVH[KLELW0HQD,19DQGPDFURSKDJH
GHSHQGHQWWUDQVHQGRWKHOLDOPLJUDWLRQ6FL6LJQDOUD
&DQFHU*HQRPH$WODV1HWZRUN&RPSUHKHQVLYHPROHFXODUSRUWUDLWVRIKXPDQ
EUHDVWWXPRXUV1DWXUH±
/LQ(<-RQHV-*/L3=KX/:KLWQH\.'0XOOHU:-HWDO3URJUHVVLRQWR
PDOLJQDQF\LQWKHSRO\RPDPLGGOH7RQFRSURWHLQPRXVHEUHDVWFDQFHUPRGHO
SURYLGHVDUHOLDEOHPRGHOIRUKXPDQGLVHDVHV$P-3DWKRO±
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
:DQJ/=KDR=0H\HU06DKD6<X0*XR$HWDO&$50PHWK\ODWHV
FKURPDWLQUHPRGHOLQJIDFWRU%$)WRHQKDQFHWXPRUSURJUHVVLRQDQG
PHWDVWDVLV&DQFHU&HOO±
+\QHV52,QWHJULQV%LGLUHFWLRQDODOORVWHULFVLJQDOLQJPDFKLQHV&HOO
±
6RWWLOH-+RFNLQJ'&)LEURQHFWLQSRO\PHUL]DWLRQUHJXODWHVWKHFRPSRVLWLRQDQG
VWDELOLW\RIH[WUDFHOOXODUPDWUL[ILEULOVDQGFHOOPDWUL[DGKHVLRQV0RO%LRO&HOO
±
5RXVVRV(7:DQJ<:\FNRII-%6HOOHUV56:DQJ:/L-HWDO0HQD
GHILFLHQF\GHOD\VWXPRUSURJUHVVLRQDQGGHFUHDVHVPHWDVWDVLVLQSRO\RPD
PLGGOH7WUDQVJHQLFPRXVHPDPPDU\WXPRUV%UHDVW&DQFHU5HV5
*HUWOHU)&RQGHHOLV-0HWDVWDVLVWXPRUFHOOVEHFRPLQJ0(1$FLQJ7UHQGV&HOO
%LRO±
+\QHV527KHH[WUDFHOOXODUPDWUL[QRWMXVWSUHWW\ILEULOV6FLHQFH
±
,\HU$.97UDQ.7*ULIILWK/:HOOV$&HOOVXUIDFHUHVWULFWLRQRI(*)5E\D
WHQDVFLQF\WRWDFWLQHQFRGHG(*)OLNHUHSHDWLVSUHIHUHQWLDOIRUPRWLOLW\UHODWHG
VLJQDOLQJ-&HOO3K\VLRO±
0LWUD$.6DZDGD.7LZDUL30XL.*ZLQ./HQJ\HO(/LJDQGLQGHSHQGHQW
DFWLYDWLRQRIF0HWE\ILEURQHFWLQDQGĮȕLQWHJULQUHJXODWHVRYDULDQFDQFHU
LQYDVLRQDQGPHWDVWDVLV2QFRJHQH±
9RJHOVWHLQ%/DQH'/HYLQH$-6XUILQJWKHSQHWZRUN1DWXUH
±
0XOOHU3$-7ULQLGDG$*7LPSVRQ30RUWRQ-3=DQLYDQ6YDQGHQ%HUJKH
39(HWDO0XWDQWSHQKDQFHV0(7WUDIILFNLQJDQGVLJQDOOLQJWRGULYHFHOO
VFDWWHULQJDQGLQYDVLRQ2QFRJHQH
+XJKHV6.2XGLQ0-7DGURV-1HLO-'HO5RVDULR$-RXJKLQ%$HWDO373%
GHSHQGHQWUHJXODWLRQRIUHFHSWRUW\URVLQHNLQDVHVLJQDOLQJE\WKHDFWLQELQGLQJ
SURWHLQ0HQD0RO%LRO&HOO
0F'RQDOG-$.HOOH\'*%URHNHOPDQQ7-5ROHRIILEURQHFWLQLQFROODJHQ
GHSRVLWLRQ)DE¶WRWKHJHODWLQELQGLQJGRPDLQRIILEURQHFWLQLQKLELWVERWK
ILEURQHFWLQDQGFROODJHQRUJDQL]DWLRQLQILEUREODVWH[WUDFHOOXODUPDWUL[-&HOO%LRO
±
&KDQ.7$VRNDQ6%.LQJ6-%R7'XERVH(6/LX:HWDO/.%ORVVLQ
PHODQRPDGLVUXSWVGLUHFWLRQDOPLJUDWLRQWRZDUGH[WUDFHOOXODUPDWUL[FXHV-&HOO
%LRO±
:\FNRII-$3DUDFULQH/RRSEHWZHHQ7XPRU&HOOVDQG0DFURSKDJHV,V5HTXLUHG
IRU7XPRU&HOO0LJUDWLRQLQ0DPPDU\7XPRUV&DQFHU5HV±
%UHGIHOGW-6/LX<3HKONH&D&RQNOLQ0:6]XOF]HZVNL-0,QPDQ'5HWDO
&RPSXWDWLRQDOVHJPHQWDWLRQRIFROODJHQILEHUVIURPVHFRQGKDUPRQLFJHQHUDWLRQ
LPDJHVRIEUHDVWFDQFHU-%LRPHG2SW
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
)LJXUHOHJHQGV
)LJXUH0HQDGULYHQKDSWRWD[LVin vitroRQ)1JUDGLHQWVLQ'DQG'LV
GHSHQGHQWXSRQLWVGLUHFWLQWHUDFWLRQZLWKĮLQWHJULQDQG)DFWLQ$6FKHPDWLF
GLDJUDPRIDPLFURIOXLGLFGHYLFHXVHGIRU'RU'KDSWRWD[LVUHSUHVHQWDWLYHLPDJHRID
)1JUDGLHQWLQDFROODJHQJHODQGDGLDJUDPGHVFULELQJWKH)0,XVHGWRTXDQWLI\
KDSWRWD[LV%([SUHVVLRQRI0HQDEXWQRW9$63RU(9/LQ09'ILEUREODVWVGULYHV
KDSWRWD[LVRQD')1JUDGLHQWȝJPODWWRSRIJUDGLHQWDVPHDVXUHGE\WKH)0,
&0HQDFHOOVKDSWRWD[ZKHQSODWHGRQD')1JUDGLHQWȝJPODWWRSRI
JUDGLHQWEXWQRWRQD/1RU91JUDGLHQWDVPHDVXUHGE\WKH)0,'0'$0%FHOOV
0HQDFHOOVSODWHGLQD'FROODJHQJHODQGVXEMHFWHGWRLQFUHDVLQJFRQFHQWUDWLRQVRI
)1DWWKHWRSRIWKHJUDGLHQW(,QKLELWLRQRIĮȕZLWK3'ȝJPOEORFNHG0HQD
GULYHQKDSWRWD[LVLQ'FROODJHQJHOVDVPHDVXUHGE\)0,ZKLOHLQKLELWLRQRIĮYȕZLWK
&LOHQJLWLGHȝ0KDGQRHIIHFW)'LDJUDPRIVWUXFWXUHRI0HQDDQGLWVGRPDLQV
LQFOXGLQJWKH/(5(5GRPDLQDQGWKH)$FWLQELQGLQJGRPDLQ)$%'HOHWLRQRIWKH
/(5(5GRPDLQDEURJDWHVWKHLQWHUDFWLRQRI0HQD0HQD,19ZLWKĮ*
0HQDǻ/(5(5DQG0HQDǻ)$%FHOOVGLGQRWKDSWRWD[LQ'FROODJHQJHOVDQGWKLVHIIHFW
ZDVLQGHSHQGHQWRIDQHIIHFWRQYHORFLW\ȝPPLQ+)RUHDFKH[SHULPHQWQ H[SHULPHQWVDWOHDVWFHOOVWUDFNHGSHUFRQGLWLRQ5HVXOWVVKRZPHDQ“6(0
VLJQLILFDQFHE\RQHZD\$129$SSS6HH)LJ6
)LJXUH0HQD,19GULYHVKDSWRWD[LVDWKLJK)1FRQFHQWUDWLRQVin vivoDQGin vitro
$0HQD,19FHOOVSODWHGLQD'FROODJHQJHODQGVXEMHFWHGWRLQFUHDVLQJ
FRQFHQWUDWLRQVRI)1DWWKHWRSRIWKHJUDGLHQW%,QKLELWLRQRIĮZLWK3'ȝJPO
EORFNHG0HQD,19GULYHQ'KDSWRWD[LVDVPHDVXUHGE\)0,ZKLOHLQKLELWLRQRIĮYȕZLWK
&LOHQJLWLGHKDGQRHIIHFW0HQD,19ǻ/(5(5RU0HQD,19ǻ)$%GLGQRWKDSWRWD[LQ
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
'Q H[SHULPHQWV•FHOOVWUDFNHGSHUFRQGLWLRQ&In vivoLQYDVLRQDVVD\LQWR
QHHGOHVLQVHUWHGLQWXPRUVJHQHUDWHGLQ12'6&,'PLFHZLWK0'$0%FHOOV
H[SUHVVLQJ&RQWURO*)30HQDRU0HQD,191HHGOHVFRQWDLQHGPJPOFROODJHQDQG
LQFUHDVLQJDPRXQWVRI)1Q PLFHSHUFRQGLWLRQ5HVXOWVVKRZPHDQ“6(06WDUV
DERYHFROXPQVUHSUHVHQWVLJQLILFDQFHUHODWLYHWRFROODJHQRQO\E\RQHZD\$129$'
5HSUHVHQWDWLYHLPDJHRID)1JUDGLHQW5KRGDPLQHODEHOHG)1VKRZQLQUHGRQ
FROODJHQILEHUVVKRZQLQZKLWHJHQHUDWHGXVLQJDPLFURVFDOHLPSODQWDEOHGHYLFH
LPSODQWHGLQWRWKHWXPRUWXPRUFHOOVODEHOHGZLWK*)3VKRZQLQJUHHQDQGYLVXDOL]HG
E\LQWUDYLWDOLPDJLQJ6FDOHEDULVȝP)0,RI(0'$0%DQG)680WXPRU
FHOOVH[SUHVVLQJGLIIHUHQW0HQDLVRIRUPVLQWKHDEVHQFHRIDGHYLFHRULQWKHSUHVHQFH
RIDGHYLFHUHOHDVLQJIOXRUHVFHQWO\ODEHOHG)1RUVLPLODUO\VL]HG'H[WUDQGDWDSRROHG•
PRYLHVIURP•PLFHSHUFRQGLWLRQ5HVXOWVVKRZPHDQ“6(0VLJQLILFDQFHE\RQHZD\
$129$SSS6HH)LJ6
)LJXUH0HQD,19LVDVVRFLDWHGZLWKSRRURXWFRPHLQKXPDQWXPRUVDQGUHTXLUHV
LWVLQWHUDFWLRQZLWKĮLQWHJULQIRUPHWDVWDVLV
.DSODQ0HLHUFXUYHVIRUVXUYLYDORIEUHDVWFDQFHUSDWLHQWVELQQHGE\TXDUWLOHVRI0HQD
$RU0HQD,19%P51$OHYHOVDVLQGLFDWHG4KDGWKHKLJKHVWH[SUHVVLRQ4WKH
ORZHVW'DWDDUHIURPEUHDVWFDQFHUFDVHVZLWK!\HDUVRIIROORZXS%5&$7&*$
GDWDVHWGDWDIURPHQWLUHSDWLHQWFRKRUWLQ)LJV6$'6LJQLILFDQFHFDOFXODWHGE\
ORJUDQN0DQWHO&R[WHVWKD]DUGUDWLRFDOFXODWHGE\ORJUDQNWHVWSTrend FDOFXODWHGE\ORJ
UDQNWHVWIRU7UHQGVHHPHWKRGV&&2;UHJUHVVLRQFDUULHGRXWWRDVVHVVWKH
UHODWLRQVKLSEHWZHHQ0HQDRU0HQD,19DQGWLPHWRGHDWKLQEUHDVWFDQFHUSDWLHQWV
SDWLHQWVZLWK\HDUIROORZXS'/RJLVWLFUHJUHVVLRQFDUULHGRXWWRDVVHVVWKH
UHODWLRQVKLSEHWZHHQ0HQDRU0HQD,19DQGVXUYLYDOLQEUHDVWFDQFHUSDWLHQWVSDWLHQWV
ZLWK\HDUIROORZXS(5HSUHVHQWDWLYHLPDJHVRI3\070079WXPRUVVWDLQHGIRU
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
0HQDUHGDQG0HQD,19JUHHQ6FDOHEDU ȝP)5HSUHVHQWDWLYHLPDJHVRI3\07
0079VWDLQHGIRU0HQD,19JUHHQDQGLQWHJULQĮUHG6DPHVFDOHDV(*
5HSUHVHQWDWLYHLPDJHIURPDZLOGW\SH3\07WXPRU)1UHG0HQD,19JUHHQDQGQXFOHL
'$3,VWDLQLQJ6FDOHEDU ȝP+&RUUHODWLRQEHWZHHQ0HQD,19DQGFROODJHQ)1
LQWHQVLW\'DWDIURPRYHUILHOGVIURP3\07PLFHHDFKGRWUHSUHVHQWVDQLQGLYLGXDO
ILHOG,5HSUHVHQWDWLYHLPDJHRIWXPRUVSRWIURPDWLVVXHPLFURDUUD\ZLWKKLJKOHYHOVRI
0HQD,19JUHHQDQG)1UHG-&RUUHODWLRQEHWZHHQ)1DQG0HQD,19VWDLQLQJLQWKH
HQWLUHSDWLHQWFRKRUW.0HQD,19H[SUHVVLRQLQEUHDVWFDQFHUSDWLHQWVFRPSDULQJ
SDWLHQWVZLWKRUZLWKRXWUHFXUUHQFHGDWDVKRZVPHDQ6(0/7DEOHVKRZLQJWKH
PHGLDQUHFXUUHQFHIUHHWLPHLQPRQWKVDQGFRUUHVSRQGLQJSYDOXHLQSDWLHQWVZLWKKLJK
YVORZ0HQD,19KLJKYVORZ)1RUKLJKYVORZ0HQD,19)16LJQLILFDQFHFDOFXODWHGE\
ORJUDQN0DQWHO&R[WHVW05HSUHVHQWDWLYHIOXRUHVFHQFHLPDJHVRI*)3SRVLWLYH
PHWDVWDVLVLQOXQJVRIPLFHZLWK&RQWURO0HQDRU0HQD,19WXPRUV6FDOHEDU ȝP1+(LPDJHVRI))3(VHFWLRQVFXWIURPWKHOXQJVRIPLFHEHDULQJ0'$0%
WXPRUVH[SUHVVLQJGLIIHUHQW0HQDLVRIRUPV6FDOHEDULVȝP2/XQJPHWDVWDWLF
LQGH[RI12'6&,'PLFHEHDULQJWXPRUVJURZQIURP0'$0%FHOOVH[SUHVVLQJ
GLIIHUHQW*)3WDJJHG0HQDLVRIRUPVDQGPHDVXULQJDWOHDVWFPLQGLDPHWHUQ•PLFH
SHUFHOOOLQH'DWDVKRZPHDQ“6(0VLJQLILFDQFHE\RQHZD\$129$S
SS6HH)LJ6DQG6
)LJXUH 0HQD,19 GULYHV KDSWRWD[LV YLD LQFUHDVHG ); VLJQDOLQJ $ 5HSUHVHQWDWLYH
LPDJHVRI&RQWURO0HQDDQG0HQD,19FHOOVSODWHGRQ)1DQGFROODJHQVWDLQHGZLWK
DQWLERGLHV WR Į DQG *)3 WR YLVXDOL]H WDJJHG *)30HQD RU *)3 0HQD,19 DQG ZLWK
SKDOORLGLQWRYLVXDOL]H)DFWLQ6FDOHEDU ȝP%0DJQLILFDWLRQRILQVHWVKRZQLQ$
6FDOHEDU ȝP&4XDQWLILFDWLRQRIFHOODUHDLQȝPRI0'$0%FHOOVH[SUHVVLQJ
GLIIHUHQWLVRIRUPVZKHQSODWHGRQFROODJHQPJPO)1ȝJPO'1XPEHURIĮ
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
SRVLWLYH DGKHVLRQV UHODWLYH WR FHOO DUHD IRU 0HQD DQG 0HQD,19 FHOOV IRU FHOOV
SODWHG RQ FROODJHQ RQO\ FROODJHQ PJPO )1 ȝJPO DQG LQ WKH SUHVHQFH RI
3'ĮIXQFWLRQEORFNLQJDQWLERG\(1XPEHURIĮSRVLWLYHDGKHVLRQVUHODWLYHWRFHOO
DUHD IRU FHOOV SODWHG RQ FROODJHQ )1 ) ,QWHQVLW\ RI Į VLJQDO LQ 0HQDSRVLWLYH
DGKHVLRQVDVFRXQWHGE\*)3SRVLWLYLW\IRU0HQDDQG0HQD,19FHOOVSODWHGRQ
FROODJHQ RQO\ FROODJHQ PJPO )1 ȝJPO DQG LQ WKH SUHVHQFH RI 3' Į
IXQFWLRQ EORFNLQJ DQWLERG\ * ,QWHQVLW\ RI Į VLJQDO LQ 0HQDSRVLWLYH DGKHVLRQV DV
FRXQWHG E\ *)3 SRVLWLYLW\ LQ FHOOV SODWHG RQ D ORZ ' ȝJPO )1 JUDGLHQW +
,QKLELWLRQ RI )$. ȝ0 GHFUHDVHG 0HQD,19GULYHQ ' KDSWRWD[LV RQ D ORZ ' ȝJPO )1 JUDGLHQW 'DWD IURP • H[SHULPHQWV ZLWK • FHOOV WUDFNHG SHU FRQGLWLRQ ,
5HSUHVHQWDWLYH LPDJHV RI &RQWURO 0HQD DQG 0HQD,19 0HQDǻ/(5(5 DQG
0HQDǻ)$%FHOOVSODWHGRQDORZ'ȝJPO)1JUDGLHQWVWDLQHGZLWKS)$.UHG
DQG 3KDOORLGLQ WR YLVXDOL]HG )DFWLQ EOXH 6FDOH EDU ȝP - 4XDQWLILFDWLRQ RI S)$.
<SRVLWLYHDGKHVLRQVLQ&RQWURO0HQDRU0HQD,19ZKLOHSODWHGRQD'ORZ
ȝJPO )1 JUDGLHQW . 0HQD,19ǻ/(5(5 RU 0HQD,19ǻ)$% FHOOV KDG GHFUHDVHG
QXPEHU RI S)$. <SRVLWLYH DGKHVLRQV RQ D ' ORZ ȝJPO )1 JUDGLHQW /
4XDQWLILFDWLRQRIS)$.<SRVLWLYHDGKHVLRQVLQ&RQWURO0HQDRU0HQD,19ZKLOH
SODWHGRQD'KLJKȝJPO)1JUDGLHQW'DWDSRROHGIURPH[SHULPHQWVZLWKDWOHDVW
FHOOV DQDO\]HG SHU FRQGLWLRQ 0 5HSUHVHQWDWLYH LPDJH RI D :% IRU Į
LPPXQRSUHFLSLWDWLRQIURP0HQDDQG0HQD,19O\VDWHVSUREHGIRU0HQDĮDQG
7XEXOLQ 1 4XDQWLILFDWLRQ RI IROG LQFUHDVH LQ 0HQD SXOOHG GRZQ LQ Į,3 Q )RU DOO
VWDLQLQJ H[SHULPHQWV GDWD SRROHG IURP DW OHDVW H[SHULPHQWV ZLWK DW OHDVW FHOOV
DQDO\]HGSHUH[SHULPHQWV5HVXOWVVKRZPHDQ“6(0VLJQLILFDQFHE\RQHZD\$129$
S S S 6WDUV DERYH GDWD VHW UHSUHVHQW VLJQLILFDQFH UHODWLYH WR
FRQWURO6HH)LJ6
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
)LJXUH0HQD,19GHSHQGHQWGLUHFWLRQDOPRWLOLW\UHTXLUHV(&0UHRUJDQL]DWLRQin
vitro$5HSUHVHQWDWLYHLPDJHVRI0'$0%FHOOVRXWOLQHGLQZKLWHDQG0HQD,19
FHOOVJUHHQLQD'ȝJPO)1JUDGLHQWUHGLQDFROODJHQJHOEOXHLQPHUJHG
LPDJHPLGGOHDQGULJKWSDQHOVVKRZJUD\VFDOHLPDJHVRI)1DQG&ROODJHQDORQH
UHVSHFWLYHO\6FDOHEDULVȝP0HQD,19VKRZHGLQFUHDVHGDFFXPXODWLRQDQG
UHRUJDQL]DWLRQRI%)1DQG&&ROODJHQLQERWKORZȝJPODQGKLJKȝJPO'
)1JUDGLHQWV'$JUDGLHQWRIUHFRPELQDQWGRPDLQVRI)1IDLOHGWRLQGXFH'
KDSWRWD[LVRI0HQD,19FHOOVDVPHDVXUHGE\WKH)0,(,QKLELWLRQRI)1
ILEULOORJHQHVLVE\LQFOXVLRQRIDN'IUDJPHQWDEODWHG0HQD,19±GHSHQGHQWKDSWRWD[LVLQ
')1JUDGLHQWVQ H[SHULPHQWV•FHOOVWUDFNHGSHUFRQGLWLRQ'HOHWLRQRIWKH)$%
DQG/(5(5UHJLRQVLQ0HQD,19DQGLQKLELWLRQRIĮȕZLWK3'EXWQRW&LOHQJLWLGH
UHGXFHGFROODJHQ)DQGFROODJHQ*DFFXPXODWLRQDWORZ)1FRQFHQWUDWLRQV'DWDIURP
•H[SHULPHQWVZLWK•FHOOVDQDO\]HGSHUFRQGLWLRQ5HVXOWVVKRZPHDQ“6(0
VLJQLILFDQFHE\RQHZD\$129$SSS6HH)LJ6
)LJXUH0HQD,19GULYHVFROODJHQUHRUJDQL]DWLRQLQWXPRUV$5HSUHVHQWDWLYHLPDJH
IURPDZLOGW\SHDQG0HQD3\07WXPRUVKRZLQJFROODJHQDVLPDJHGE\6+*JUD\
DQGQXFOHL'$3,VWDLQLQJ6FDOHEDU ȝP%4XDQWLILFDWLRQRIFROODJHQVLJQDODV
PHDVXUHGE\6+*VLJQDOLQZLOGW\SHDQG0HQD3\07WXPRUV'DWDIURPRYHU
ILHOGVIURPPLFHIRUZLOGW\SHPLFHDQGPLFHIRU0HQDHDFKGRWUHSUHVHQWVDQ
LQGLYLGXDOILHOG&5HSUHVHQWDWLYHLPDJHIURPDZLOGW\SH3\07WXPRUVKRZLQJFROODJHQ
DVLPDJHGE\6+*JUD\DQG0HQD,19JUHHQ6FDOHEDU ȝP'&RUUHODWLRQ
EHWZHHQ0HQD,19DQGFROODJHQLQWHQVLW\'DWDIURPRYHUILHOGVIURPPLFHHDFKGRW
UHSUHVHQWVDQLQGLYLGXDOILHOG(5HSUHVHQWDWLYHLPDJHVVKRZLQJFROODJHQE\6+*IURP
DEUHDVWFDQFHUSDWLHQWVDPSOHVZLWKKLJKRUORZ0HQD,19)&ROODJHQLQWHQVLW\LQ
SDWLHQWVZLWKKLJKRUORZ0HQD,19H[SUHVVLRQ*5HSUHVHQWDWLYHLPDJHVRIFROODJHQ
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
RUJDQL]DWLRQJUD\RI&RQWURO0HQDDQG0HQD,19[HQRJUDIWWXPRUVWDNHQE\
LQWUDYLWDOLPDJLQJ6FDOHEDULVȝP+5HSUHVHQWDWLYHGLDJUDPRIDQJOHXVHGWR
PHDVXUHWKHRULHQWDWLRQRILQGLYLGXDOFROODJHQILEHUVUHODWLYHWRWKHHGJHRIWKHWXPRUV
3ORWWHGGLVWULEXWLRQVRIFROODJHQILEHURULHQWDWLRQUHODWLYHWRWXPRUVHGJHFRPSDULQJ
&RQWURO0HQDDQG0HQD,19H[SUHVVLQJ+0'$0%DQG+680FHOOV'DWD
SRROHGIURP•LPDJHVIURP•PLFHSHUFRQGLWLRQ5HVXOWVVKRZPHDQ“6(0
VLJQLILFDQFHE\WWHVWSSS6HH)LJ6
)LJXUH6XPPDU\GLDJUDP)1OHYHOVDUHKLJKDURXQGEORRGYHVVHOVDQGDWLQYDVLYH
HGJHVLQWXPRUV([SUHVVLRQRI0HQDLQWXPRUFHOOVDOORZVFHOOVWRKDSWRWD[RQORZ
JUDGLHQWVRI)1YLDLWVZHDNDVVRFLDWLRQZLWKĮDQGLQFUHDVHG);VLJQDOLQJ([SUHVVLRQ
RI0HQD,19DOORZVFHOOVWRKDSWRWD[RQERWKORZDQGKLJK)1JUDGLHQWVYLDLQFUHDVHG
DVVRFLDWLRQZLWKĮOHDGLQJWRLQFUHDVHG);QXPEHUDQG)$.VLJQDOLQJDW);VDVZHOO
DVWKURXJK(&0UHRUJDQL]DWLRQ
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on January 25, 2016; DOI: 10.1158/2159-8290.CD-15-1183
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Tumor cell-driven extracellular matrix remodeling enables
haptotaxis during metastatic progression
Madeleine J. Oudin, Oliver Jonas, Tatsiana Kosciuk, et al.
Cancer Discov Published OnlineFirst January 25, 2016.
Updated version
Supplementary
Material
Author
Manuscript
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
doi:10.1158/2159-8290.CD-15-1183
Access the most recent supplemental material at:
http://cancerdiscovery.aacrjournals.org/content/suppl/2016/01/23/2159-8290.CD-15-1183.DC1.html
Author manuscripts have been peer reviewed and accepted for publication but have not yet been
edited.
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at pubs@aacr.org.
To request permission to re-use all or part of this article, contact the AACR Publications
Department at permissions@aacr.org.
Downloaded from cancerdiscovery.aacrjournals.org on March 22, 2016. © 2016 American Association for Cancer
Research.
Download