Aberrant glycosylation promotes lung cancer metastasis

Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
$EHUUDQW JO\FRV\ODWLRQ SURPRWHV OXQJ FDQFHU PHWDVWDVLV WKURXJK DGKHVLRQ WR JDOHFWLQV LQ
WKHPHWDVWDWLFQLFKH
1DWKDQ(5HWLFNHU)O\QQDQG6DQJHHWD1%KDWLD
'DYLG+.RFK,QVWLWXWHIRU,QWHJUDWLYH&DQFHU5HVHDUFK0DVVDFKXVHWWV,QVWLWXWHRI
7HFKQRORJ\&DPEULGJH0DVVDFKXVHWWV86$
+DUYDUG0,7'LYLVLRQRI+HDOWK6FLHQFHVDQG7HFKQRORJ\0DVVDFKXVHWWV,QVWLWXWHRI
7HFKQRORJ\&DPEULGJH0DVVDFKXVHWWV86$
+RZDUG+XJKHV0HGLFDO,QVWLWXWH&DPEULGJH0DVVDFKXVHWWV86$
'LYLVLRQRI0HGLFLQH%ULJKDPDQG:RPHQ¶V+RVSLWDO%RVWRQ0DVVDFKXVHWWV
86$
'HSDUWPHQWRI(OHFWULFDO(QJLQHHULQJDQG&RPSXWHU6FLHQFH0DVVDFKXVHWWV,QVWLWXWHRI
7HFKQRORJ\&DPEULGJH0DVVDFKXVHWWV86$
7RZKRPFRUUHVSRQGHQFHVKRXOGEHVHQWVEKDWLD#PLWHGX
5XQQLQJ7LWOHSt6galnac4Gcnt3DQGJDOHFWLQVDVGULYHUVRI16&/&PHWDVWDVLV
.H\:RUGV16&/&PHWDVWDVLVJO\FRV\ODWLRQJO\FRV\OWUDQVIHUDVHJDOHFWLQ,/7KRPVHQ
)ULHGHQUHLFK$QWLJHQSt6galnac4Gcnt3WXPRUPLFURHQYLURQPHQW
7KLVZRUNZDVVXSSRUWHGLQSDUWE\WKH.RFK,QVWLWXWH6XSSRUWFRUH*UDQW3&$IURP
WKH1DWLRQDO&DQFHU,QVWLWXWH15HWLFNHU)O\QQLVVXSSRUWHGE\D/XGZLJ&HQWHUIRU0ROHFXODU
2QFRORJ\*UDGXDWH)HOORZVKLS6%KDWLDLVDQ++0,LQYHVWLJDWRU7KLVZRUNZDVVXSSRUWHGE\
D6WDQG8S7R&DQFHU'UHDP7HDP7UDQVODWLRQDO5HVHDUFK*UDQW*UDQW1XPEHU68&$$&5
'76WDQG8S7R&DQFHULVDSURJUDPRIWKH(QWHUWDLQPHQW,QGXVWU\)RXQGDWLRQ
DGPLQLVWHUHGE\WKH$PHULFDQ$VVRFLDWLRQIRU&DQFHU5HVHDUFKDQGWKH.RFK&7&3URMHFW´
6%KDWLDFRQWDFWLQIRUPDWLRQ
0DVVDFKXVHWWV$YH
%XLOGLQJ
&DPEULGJH0$
VEKDWLD#PLWHGX
SKRQH
ID[
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
7KHDXWKRUVKDYHQRFRQIOLFWVRILQWHUHVWWRGLVFORVH
:RUG&RXQWLQFOXGLQJ0HWKRGV
:RUG&RXQWH[FOXGLQJ0HWKRGV
)LJXUH&RXQW
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
$EVWUDFW
0HWDVWDVLVLVWKHOHDGLQJFDXVHRIFDQFHUDVVRFLDWHGGHDWKV:KLOHGLVVHPLQDWLRQRIWXPRUFHOOV
OLNHO\RFFXUVHDUO\LQWXPRULJHQHVLVWKHFRQVWLWXHQWVRIWKHPLFURHQYLURQPHQWSOD\HVVHQWLDOUDWH
OLPLWLQJ UROHV LQ GHWHUPLQLQJ ZKHWKHU WKHVH FHOOV ZLOO IRUP FOLQLFDOO\UHOHYDQW WXPRUV 5HFHQW
VWXGLHV KDYH XQFRYHUHG PDQ\ PROHFXODU IDFWRUV WKDW FRQWULEXWH WR HVWDEOLVKPHQW RI D SUR
WXPRULJHQLF PHWDVWDWLF QLFKH +HUH ZH GHPRQVWUDWH WKDW JDOHFWLQ ZKRVH H[SUHVVLRQ KDV
FOLQLFDO DVVRFLDWLRQV ZLWK DGYDQFHG PDOLJQDQF\ DQG SRRU RXWFRPH FRQWULEXWHV WR PHWDVWDWLF
QLFKH IRUPDWLRQ E\ ELQGLQJ WR FDUERK\GUDWHV RQ PHWDVWDWLF FHOOV :H VKRZ WKDW JDOHFWLQ LV
H[SUHVVHG HDUO\ GXULQJ WXPRULJHQHVLV E\ ERWK &'E*U DQG &'E/\&KL OHXNRF\WHV
7XPRUVPRELOL]HWKHVHP\HORLGSRSXODWLRQVWKURXJKVHFUHWLRQRIVROXEOHIDFWRUVLQFOXGLQJ,/
:H ILQG WKDW PHWDVWDWLF FDQFHU FHOOV H[KLELW HOHYDWHG SUHVHQWDWLRQ RI WKH RQFRIHWDO JDOHFWLQ
FDUERK\GUDWHOLJDQGWKH7KRPVHQ)ULHGHQUHLFK$QWLJHQRQWKHLUVXUIDFHVDVDUHVXOWRIDOWHUHG
&*Q7DQG6W*DO1$F,9JO\FRV\OWUDQVIHUDVHDFWLYLW\WKDWLQKLELWVIXUWKHUJO\FRV\ODWLRQRIWKLV
FDUERK\GUDWHPRWLIDQGSURPRWHVPHWDVWDVLV
6LJQLILFDQFH
:KLOH FOLQLFDO REVHUYDWLRQV RI HOHYDWHG VHUXP JDOHFWLQ OHYHOV DQG DOWHUHG JO\FRV\ODWLRQ KDYH
EHHQDVVRFLDWHGZLWKPDOLJQDQF\ZHLGHQWLI\QRYHOUROHVIRUJO\FRV\OWUDQVIHUDVHVLQSURPRWLQJ
DGKHVLRQ WR JDOHFWLQV LQ WKH PHWDVWDWLF QLFKH 7KLV LGHQWLILFDWLRQ RI D F\WRNLQHOHXNRF\WH
JO\FRV\ODWLRQ D[LV LQ PHWDVWDVLV SURYLGHV PHFKDQLVWLF H[SODQDWLRQV IRU FOLQLFDO DVVRFLDWLRQV
EHWZHHQPDOLJQDQF\DQGDEHUUDQWJO\FRV\ODWLRQ
,QWURGXFWLRQ
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
*DOHFWLQVDUHDIDPLO\RIȕJDODFWRVLGHELQGLQJOHFWLQVZKRVHSOHLRWURSLFUROHVLQWXPRULJHQHVLV
KDYH EHHQ UHSRUWHG WR UDQJH IURP LQWHUDFWLRQV ZLWK RQFRSURWHLQV WR LPPXQRPRGXODWLRQ WR
SURPRWLRQ RI DQJLRJHQHVLV WR HOLFLWLQJ DQWLDSRSWRWLF HIIHFWV WKURXJK %&/ KRPRORJLHV 'HVSLWH WKH IDFW WKDW JDOHFWLQV ODFN D VLJQDO VHTXHQFH RU WUDQVPHPEUDQH GRPDLQ WKH\ FDQ EH
LQFRUSRUDWHG LQWR WKH H[WUDFHOOXODU PDWUL[ (&0 VHFUHWHG DV VROXEOH IDFWRUV LQWR FLUFXODWLRQ
SUHVHQWHG RQ FHOO VXUIDFHV RU DFW LQWUDFHOOXODUO\ ,Q SDUWLFXODU WKH SUHVHQFH RI JDOHFWLQ DV DQ
H[WUDFHOOXODU PROHFXOH FLUFXODWLQJ LQ VHUXP RU ORFDOL]HG RQ FHOOV ZLWKLQ WLVVXHV KDV EHHQ
DVVRFLDWHG ZLWK PHWDVWDVLV (OHYDWHG OHYHOV RI JDOHFWLQ KDYH EHHQ UHSRUWHG LQ SDWLHQWV ZLWK
DGYDQFHGVWDJHVRIDYDULHW\RIFDQFHUVDQGWUHDWPHQWRIPLFHEHDULQJ[HQRJUDIWWXPRUV
ZLWKDWUXQFDWHGIRUPRIWKHSURWHLQWKDWDFWVDVDFRPSHWLWLYHELQGLQJLQKLELWRUKDVEHHQUHSRUWHG
WR UHGXFH LQFLGHQFHV RI PHWDVWDVHV ,Q SDUWLFXODU JDOHFWLQ DQG JDOHFWLQ FRQWDLQ
FDUERK\GUDWHUHFRJQLWLRQGRPDLQV&5'VWKDWH[KLELWVSHFLILFDIILQLWLHVIRUWKHRQFRIHWDOFRUH
O-OLQNHG GLVDFFKDULGH WHUPHG WKH 7KRPVHQ)ULHGHQUHLFK$QWLJHQ7$QWLJHQ :KLOH WKLV
PXFLQW\SH JO\FDQ PRWLI IRUPV WKH FRUH VWUXFWXUH RI PRVW OJO\FDQV *DOȕ*DO1$FĮ
6HU7KUH[SRVXUHRIWKHGLVDFFKDULGHIRUPLVJHQHUDOO\RFFOXGHGE\IXUWKHUJO\FRV\ODWLRQ,WLV
HVWLPDWHG KRZHYHU WKDW WKH FHOOV LQ DV PDQ\ DV RI FDUFLQRPDV H[SUHVV WKLV GLVDFFKDULGH
ZLWKRXWIXUWKHUJO\FRV\ODWLRQ1RQHWKHOHVVWKHPHFKDQLVPVE\ZKLFKWKLVFRQVHUYHGJO\FDQ
PRWLILVUHJXODWHGE\FDQFHUFHOOVDORQJZLWKWKHSUHFLVHUROHWKDWLWSOD\VLQWXPRULJHQHVLVDQG
PHWDVWDVLVDUHSRRUO\XQGHUVWRRG
5HFHQWO\ WKH UROH WKDW G\QDPLF UHPRGHOLQJ RI WKH WXPRU PLFURHQYLURQPHQW SOD\V LQ WKH
SURPRWLRQ RI PHWDVWDVLV KDV EHHQ SDUWLDOO\ HOXFLGDWHG 6ROXEOH IDFWRUV UHOHDVHG IURP
WXPRUVDFWWRPRELOL]HERQHPDUURZGHULYHGFHOOV%0'&VDQGLQSDUWLFXODUP\HORLGFHOOVLQWR
WKHSHULSKHUDOEORRGDQGUHFUXLWWKHPWRWKHHDUO\PHWDVWDWLFQLFKHDVLWHZKHUHPHWDVWDVHVKDYH
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
\HW WR UHDFK D GHWHFWDEOH VL]H )UHTXHQWO\ WKHVH P\HORLG FHOOV FRQVLVWRI &'E FHOOV WKDW DUH
/\&KLRU*U7KHIRUPHUSRSXODWLRQW\SLFDOO\FRQVLVWVRILQIODPPDWRU\PRQRF\WHVZKLOHWKH
ODWWHUPD\UHSUHVHQWQHXWURSKLOVRUDKHWHURJHQHRXVSRRUO\GLIIHUHQWLDWHGJUDQXORF\WHSRSXODWLRQ
WKDW H[KLELWV VXSSUHVVRU IXQFWLRQV VRPHWLPHV UHIHUUHG WR DV P\HORLGGHULYHG VXSSUHVVRU FHOOV
0'6&V&URVVWDONEHWZHHQSULPDU\RUPHWDVWDWLFWXPRUVDQGWKHVHRURWKHUQRQSDUHQFK\PDO
SRSXODWLRQV LQGXFH WKH VHFUHWLRQ RI IDFWRUV LQ DXWRFULQH SDUDFULQH DQG HQGRFULQH IDVKLRQV LQ D
PDQQHU WKDW SURPRWHV UHPRGHOLQJ RI WKH SULPDU\ WXPRU ERQH PDUURZ DQG PHWDVWDVHV WR
SURPRWH PDOLJQDQW SURJUHVVLRQ $PRQJ WKH PROHFXOHV WKDW DUH LQYROYHG LQ PHWDVWDWLF QLFKH
IRUPDWLRQ PDQ\ DUH H[WUDFHOOXODU PDWUL[ (&0 PROHFXOHV DQG WKHLU PRGLILHUV LQFOXGLQJ
ILEURQHFWLQFROODJHQ,9WHQDVFLQFRVWHRSRQWLQSHULRVWLQYHUVLFDQO\V\OR[LGDVH/2;DQGD
YDULHW\ RI PDWUL[ PHWDOORSURWHLQDVHV 003V 7KH UROH RI FHOOVXUIDFH JDOHFWLQV
KRZHYHUDVFRPSRQHQWVRIWKHHDUO\PHWDVWDWLFQLFKHUHPDLQVWREHLQYHVWLJDWHG
$OWHUDWLRQVLQJHQHH[SUHVVLRQF\WRNLQHVHFUHWLRQDQG(&0DQGVROXEOHIDFWRUSURGXFWLRQKDYH
DOO EHHQ DVVRFLDWHG ZLWK LPSURYHG LQWHUDFWLRQV EHWZHHQ PHWDVWDWLF FHOOV DQG VSHFLILF
PLFURHQYLURQPHQWV 7R GHWHUPLQH WKH LQYROYHPHQW RI JDOHFWLQ ZLWKLQ
WKH PHWDVWDWLF QLFKH DQG RI DEHUUDQW JO\FRV\ODWLRQ RI FDQFHU FHOODVVRFLDWHG VXUIDFH
JO\FRSURWHLQVZHXVHGDPRXVHPRGHORIOXQJDGHQRFDUFLQRPDPHWDVWDVLV3UHYLRXVO\
ZH XVHG RXU (&0 PLFURDUUD\ SODWIRUP WR LGHQWLI\ DGKHVLRQ OLJDQGV DVVRFLDWHG ZLWK PHWDVWDVLV
DQGLGHQWLILHGJDOHFWLQDQGDVH[KLELWLQJSUHIHUHQWLDODGKHVLRQWRPHWDVWDWLFFHOOV+HUH
ZH LGHQWLI\ JDOHFWLQ DV D FRPSRQHQW RI WKH PHWDVWDWLF QLFKH WKURXJK H[SUHVVLRQ RQ P\HORLG
FHOOV :H ILQG WKDW FKDQJHV LQ JO\FRV\ODWLRQ E\ WKH FDQFHU FHOOV PHGLDWH WKHLU LQFUHDVHG
LQWHUDFWLRQVZLWKJDOHFWLQVDQGWKDWWKHVHDOWHUDWLRQVSOD\HVVHQWLDOUROHVLQFRQIHUULQJPHWDVWDWLF
SRWHQWLDO,QSDUWLFXODUZHILQGWKDWWKHXSUHJXODWLRQRIWKHVLDO\OWUDQVIHUDVH6W*DO1$F,9DQG
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
GRZQUHJXODWLRQ RI WKH FRUH NDFHW\OJOXFRVDPLQ\OWUDQVIHUDVH &*Q7 Gcnt3 DUH NH\
PHGLDWRUV RI WKH FKDQJHV LQ JO\FRV\ODWLRQ WKDW SRWHQWLDWH PHWDVWDWLF SURFOLYLW\ E\ SUHVHUYLQJ
SUHVHQWDWLRQRIWKH7$QWLJHQ
5HVXOWV
7XPRUEHDULQJPLFHH[KLELWHOHYDWHGOHYHOVRIJDOHFWLQLQWKHHDUO\PHWDVWDWLFQLFKH
7R GHWHUPLQH WKH UROH RI JDOHFWLQ LQ PHWDVWDVLV ZH XVHG D JHQHWLF PRXVH PRGHO RI OXQJ
DGHQRFDUFLQRPDPHWDVWDVLVKrasLSL-G12D/+;p53flox/floxIURPZKLFKFHOOOLQHVUHSUHVHQWLQJGLVWLQFW
VWDJHV RI PHWDVWDWLF SURJUHVVLRQ ZHUH GHULYHG )ROORZLQJ &UHPHGLDWHG UHFRPELQDWLRQ
DQG WKH GHYHORSPHQW RI DXWRFKWKRQRXV OXQJ WXPRUV DQG PHWDVWDVHV OLQHV ZHUH JHQHUDWHG IURP
SULPDU\WXPRUVWKDWGLGQRWJLYHULVHWRPHWDVWDVHV7QRQ0HWGLGJLYHULVHWRPHWDVWDVHV70HW
O\PSK QRGH PHWDVWDVHV 1 DQG OLYHU PHWDVWDVHV 0 3UHYLRXVO\ ZH IRXQG WKDW LQFUHDVLQJO\
PHWDVWDWLF OLQHV H[KLELWHG LQFUHDVHG DGKHVLRQ WR JDOHFWLQ in vitro )LJ $ $V (&0
PROHFXOHV DQG WKHLU PRGLILFDWLRQ E\ %0'&V SOD\ LPSRUWDQW UROHV LQ WKH GHYHORSPHQW RI WKH
PHWDVWDWLF QLFKH ZH DVNHG ZKHWKHU JDOHFWLQ KDV D UROH LQ LWV HVWDEOLVKPHQW /LYHUV ZHUH
KDUYHVWHGIURPWXPRUEHDULQJPLFHSULRUWRWKHGHWHFWLRQRIRYHUWPHWDVWDVHVDQGH[DPLQHGIRU
DFFXPXODWLRQRIJDOHFWLQ:HVWHUQEORWDQDO\VLVRIOLYHUSURWHLQO\VDWHVUHYHDOHGDQHOHYDWLRQLQ
JDOHFWLQLQWKHOLYHUVRIWXPRUEHDULQJPLFHFRPSDUHGWRQDwYHPLFH)LJ%7RLGHQWLI\WKH
VRXUFH RI WKLV JDOHFWLQ ZH H[DPLQHG ERWK WKH WXPRU FHOOV DQG UHFUXLWHG OHXNRF\WHV *HQH
H[SUHVVLRQ PLFURDUUD\ DQG ZHVWHUQ EORW DQDO\VHV RI JDOHFWLQ LQ WKH WXPRU FHOO OLQHV UHYHDOHG
HTXLYDOHQWH[SUHVVLRQRIWKHPROHFXOHVDFURVVDOOIRXUFODVVHVRIOLQHVLQGLFDWLQJWKDWJDOHFWLQ
H[SUHVVLRQ E\ WXPRUV LV QRW GLIIHUHQWLDOO\ UHJXODWHG GXULQJ WXPRU SURJUHVVLRQ 6XSSOHPHQWDU\
)LJ $ % ,Q OLJKW RI UHFHQW UHSRUWV RI P\HORLGGHULYHG SRSXODWLRQV VXSSRUWLQJ RXWJURZWK RI
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
PHWDVWDWLF WXPRUV KRZHYHU ZH DVNHG ZKHWKHU VXFK SRSXODWLRQV PLJKW DOVR EH SUHVHQWLQJ
JDOHFWLQ ZLWKLQ WKH HDUO\ PHWDVWDWLF QLFKH 6WDLQLQJ IRU PDFURSKDJHV DQG JDOHFWLQ LQ OLYHU
VHFWLRQVRIPLFHEHDULQJHLWKHU7QRQ0HWRU0WXPRUVEXWQRGHWHFWDEOHRYHUWPHWDVWDVHVUHYHDOHG
FRORFDOL]DWLRQ RI JDOHFWLQ ZLWK )SRV PDFURSKDJHV )LJ & :KLOH JDOHFWLQSRV
PDFURSKDJHVDUHSUHVHQWLQWKHOLYHUVRIWXPRUIUHHPLFHDVZHOOWKHLUQXPEHUVDUHWZRWRIRXU
WLPHVORZHUWKDQREVHUYHGLQWXPRUEHDULQJPLFH)LJ&)XUWKHUPRUHLPPXQRVWDLQLQJRIWKH
HQJUDIWHGWXPRUVDOVRUHYHDOHGDGHQVHPDFURSKDJHLQILOWUDWLRQWKDWFRORFDOL]HGZLWKJDOHFWLQ
VWDLQLQJ 6XSSOHPHQWDU\ )LJ & :KLOH RWKHU VRXUFHV RI JDOHFWLQ FRXOG LQFOXGH VHFUHWLRQ
GLUHFWO\ E\ WKH WXPRUV WKHVH ILQGLQJV VXJJHVWHG WKH SRVVLELOLW\ WKDW OHXNRF\WHV PLJKW EH
H[SUHVVLQJJDOHFWLQWKHPVHOYHV
7XPRUEHDULQJ PLFH H[KLELW FLUFXODWLQJ &'EJDOHFWLQ P\HORLG FHOOV HDUO\ LQ
WXPRULJHQHVLV
*LYHQ RXU REVHUYDWLRQ RI JDOHFWLQH[SUHVVLQJPDFURSKDJHV LQ WKH PHWDVWDWLF QLFKH ZH DVNHG
ZKHWKHU WXPRUV LQGXFH WKH PRELOL]DWLRQ RI OHXNRF\WHV H[SUHVVLQJ JDOHFWLQ :H KDUYHVWHG
SHULSKHUDOEORRGIURPQDwYHPLFHRUPLFHEHDULQJWXPRUVJHQHUDWHGE\HLWKHU7QRQ0HWRU0OLQHV
6XSSOHPHQWDU\)LJ$&DQGDQDO\]HGLWIRUWKHSUHVHQFHRIJDOHFWLQDQG&'EP\HORLG
FHOOV $QDO\VLV RI SHULSKHUDO EORRG FHOOV E\ IORZ F\WRPHWU\ UHYHDOHG D PDUNHG LQFUHDVH LQ
JDOHFWLQ DQG &'E OHXNRF\WHV LQ WKH FLUFXODWLRQ RI PLFH EHDULQJ ERWK WKH 0 DQG 7QRQ0HW
WXPRUV )LJ $& DQG 6XSSOHPHQWDU\ )LJ ') &RQVLVWHQW ZLWK WKH ILQGLQJV RI JDOHFWLQ
PDFURSKDJHV LQ WKH OLYHU WKHUH ZDV DQ HQULFKPHQW LQ JDOHFWLQ&'E GRXEOHSRVLWLYH
SRSXODWLRQVLQWKHEORRGRIWXPRUEHDULQJPLFH)LJ$',QDJUHHPHQWZLWKSUHYLRXVUHSRUWV
RI LQFUHDVHV LQ LQIODPPDWRU\ P\HORLG FHOOV LQ WXPRUEHDULQJ PLFH WKHUH ZDV D VLJQLILFDQW
LQFUHDVH LQ WKH IUHTXHQF\ RI &'E*U DQG &'E/\&KL SRSXODWLRQV )LJ () DQG
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
6XSSOHPHQWDU\ )LJ *+ ZKLFK PD\ UHSUHVHQW QHXWURSKLOV RU 0'6&V DQG LQIODPPDWRU\
PRQRF\WHV,0VUHVSHFWLYHO\ZLWKWKHODWWHUSRSXODWLRQEHLQJ&'6XSSOHPHQWDU\)LJ,
&RPSDULVRQV RI WKH JDOHFWLQ OHYHOV RQ WKH &'E/\&KL DQG &'E/\&OR SRSXODWLRQV
UHYHDO WKUHH WR IRXUIROG KLJKHU H[SUHVVLRQ RQ WKH HQULFKHG SRSXODWLRQ )LJ * ,Q FRQWUDVW
JDOHFWLQOHYHOVRQWKHYDULRXVVXESRSXODWLRQVRI&'EOHXNRF\WHVZHUHQRWDIIHFWHGE\WKH
SUHVHQFHRIWXPRUV6XSSOHPHQWDU\)LJ$&VXJJHVWLQJWKDWWKHUHLVLQFUHDVHGPRELOL]DWLRQRI
PXOWLSOH JDOHFWLQ SRSXODWLRQV LQWR WKH FLUFXODWLRQ UDWKHU WKDQ LQFUHDVHG H[SUHVVLRQ RQ
LQGLYLGXDOOHXNRF\WHV
*DOHFWLQKDVEHHQUHSRUWHGWRELQGDYDULHW\RIPDFURSKDJHFHOOVXUIDFHJO\FRSURWHLQV7R
GHWHUPLQHZKHWKHUWKHJDOHFWLQLVERXQGWROHXNRF\WHVWKURXJKFDUERK\GUDWHLQWHUDFWLRQVRULV
SUHVHQWHG LQ D PDQQHU WKDW VWLOO SHUPLWV LQWHUDFWLRQV ZLWK WKH FDUERK\GUDWH UHFRJQLWLRQ GRPDLQ
&5'ZHLVRODWHG&'EFHOOVIURPWKHEORRGRIWXPRUEHDULQJPLFHDQGLQFXEDWHGWKHPLQ
WKH SUHVHQFH RI D FRPSHWLWLYH JDOHFWLQ OLJDQG ODFWRVH RU D FRQWURO VXFURVH 6XEVHTXHQW
DQDO\VLV UHYHDOHG QR VLJQLILFDQW GLIIHUHQFH LQ JDOHFWLQ ELQGLQJ VXJJHVWLQJ WKDW JDOHFWLQ LV
SUHVHQWHG RQ WKH VXUIDFHV RI WKHVH SRSXODWLRQV LQ D FDUERK\GUDWHLQGHSHQGHQW PDQQHU WKXV
SUHVHUYLQJWKHDYDLODELOLW\RIWKH&5'WRLQWHUDFWZLWKRWKHUJO\FDQOLJDQGV6XSSOHPHQWDU\)LJ
7DNHQ WRJHWKHU WKHVH GDWD VXSSRUW WKH K\SRWKHVLV WKDW WKH SUHVHQFH RI WXPRUV LQGXFHV WKH
PRELOL]DWLRQRI&'EJDOHFWLQP\HORLGGHULYHGFHOOVWRWKHEORRGDQGWKDWWKHVHOHXNRF\WHV
SUHVHQWJDOHFWLQLQDPDQQHUWKDWSHUPLWVDGGLWLRQDOFDUERK\GUDWHLQWHUDFWLRQVZLWKWKHOHFWLQ
7XPRUGHULYHG LQWHUOHXNLQ PRELOL]HV &'EJDOHFWLQ OHXNRF\WHV LQWR WKH SHULSKHUDO
EORRG
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
7XPRUGHULYHG VROXEOH IDFWRUV IUHTXHQWO\ LQGXFH WKH PRELOL]DWLRQ DQG UHFUXLWPHQW RI SUR
PHWDVWDWLF &'E SRSXODWLRQV 7R GHWHUPLQH ZKHWKHU WXPRUVHFUHWHG
IDFWRUV ZHUH UHVSRQVLEOH IRU WKH REVHUYHG PRELOL]DWLRQ RI &'EJDOHFWLQ SRSXODWLRQV ZH
FROOHFWHGFRQGLWLRQHGPHGLD&0IURPWKHFHOOOLQHVDQGLQMHFWHGLWLQWRWKHFLUFXODWLRQRIZLOG
W\SH PLFH 7ZR KRXUV IROORZLQJ LQMHFWLRQ RI WKH &0 ZH KDUYHVWHG SHULSKHUDO EORRG DQG
DQDO\]HG WKH SRSXODWLRQV E\ IORZ F\WRPHWU\ 7KLV DQDO\VLV UHYHDOHG SURQRXQFHG LQFUHDVHV LQ
FLUFXODWLQJ &'EJDOHFWLQ OHXNRF\WHV DQG UHOHYDQW VXESRSXODWLRQV )LJ $ DQG
6XSSOHPHQWDU\)LJ$'$JDLQGLIIHUHQFHVLQWKHOHYHORIJDOHFWLQH[SUHVVLRQRQLQGLYLGXDO
FHOOVZHUHQRWGHWHFWDEOHLQWKHSHULSKHUDOEORRGEHWZHHQWKHWZRFRQGLWLRQV6XSSOHPHQWDU\)LJ
(VXJJHVWLQJWKDWWKHHOHYDWHGJDOHFWLQOHYHOVZHUHGXHWRLQFUHDVHGPRELOL]DWLRQRIJDOHFWLQ
SRSXODWLRQVUDWKHUWKDQLQFUHDVHGH[SUHVVLRQRIJDOHFWLQE\WKHPRELOL]HGOHXNRF\WHV
6ROXEOH JDOHFWLQ KDV EHHQ UHSRUWHG WR DFW DV D FKHPRDWWUDFWDQW IRU PDFURSKDJHV PRQRF\WHV
DQGQHXWURSKLOV7KHJDOHFWLQWKDWZHKDYHREVHUYHGKDVSULPDULO\EHHQSUHVHQWHGRQ
WKHVXUIDFHVRIOHXNRF\WHV)LJ&DQG$VWKHWXPRUVDOVRVHFUHWHJDOHFWLQKRZHYHUZH
DVNHGZKHWKHUJDOHFWLQLWVHOILVFDSDEOHRILQGXFLQJWKHUDSLGPRELOL]DWLRQRI&'EJDOHFWLQ
OHXNRF\WHV,QMHFWLRQVRIUHFRPELQDQWPXULQHJDOHFWLQLQFRQWUROPHGLXPKRZHYHUIDLOHGWR
HOLFLW DQ\ LQFUHDVHG PRELOL]DWLRQ RI WKH &'EJDOHFWLQ SRSXODWLRQV )LJ $ DQG
6XSSOHPHQWDU\)LJ1RQHWKHOHVVLWLVSRVVLEOHWKDWJDOHFWLQPLJKWDFWLQFRPELQDWLRQZLWK
RWKHU VHFUHWHG IDFWRUV WR PRELOL]H WKHVH SRSXODWLRQV 7R GHWHUPLQH ZKHWKHU JDOHFWLQ LV
QHFHVVDU\WRLQGXFHPRELOL]DWLRQZHNQRFNHGGRZQJDOHFWLQLQWKH0OLQHXVLQJVKRUWKDLUSLQV
6XSSOHPHQWDU\)LJ$:KLOHWKLVNQRFNGRZQUHGXFHGWKH&0FRQFHQWUDWLRQVRIJDOHFWLQE\
DSSUR[LPDWHO\ WKHUH ZDV QR REVHUYDEOH GLIIHUHQFH LQ WKH PRELOL]DWLRQ RI DQ\ RI WKH
JDOHFWLQOHXNRF\WHVRUWKHLUVXUIDFHOHYHOVRIJDOHFWLQ)LJ%DQG6XSSOHPHQWDU\)LJ%
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
))XUWKHUPRUHPLFHEHDULQJWXPRUVIRUPHGE\WKHNQRFNGRZQOLQHVH[KLELWHGQRGLIIHUHQFHVLQ
FLUFXODWLQJJDOHFWLQOHXNRF\WHVLQWKHSHULSKHUDOEORRGRUWKHQXPEHURIJDOHFWLQSRV)SRV
PDFURSKDJHVLQWKHLUOLYHUVGDWDQRWVKRZQDQG6XSSOHPHQWDU\)LJ*7DNHQWRJHWKHUWKHVH
GDWD VXJJHVW WKDW WXPRUGHULYHG JDOHFWLQ KDV QR HIIHFW RQ WKH PRELOL]DWLRQ RI JDOHFWLQ
OHXNRF\WHV RU WKHLU SUHVHQWDWLRQ RI JDOHFWLQ QRU GRHV LW DFW LQ DQ DXWRFULQH IDVKLRQ RQ WXPRU
FHOOVWRLQGXFHVHFUHWLRQRIRWKHULQIODPPDWRU\F\WRNLQHV
:KLOHJDOHFWLQGRHVQRWDIIHFWPRELOL]DWLRQRIOHXNRF\WHVZHDVNHGZKHWKHULWVH[SUHVVLRQE\
PHWDVWDWLF FHOOV LV QHFHVVDU\ IRU VHHGLQJ RU FRORQL]DWLRQ 7R GHWHUPLQH ZKHWKHU WXPRUGHULYHG
JDOHFWLQDFWVLQDFHOODXWRQRPRXVPDQQHUWRVXSSRUWJURZWKRIGLVVHPLQDWHGWXPRUFHOOVZH
SHUIRUPHG H[SHULPHQWDO PHWDVWDVLV DVVD\V ZLWK WKH JDOHFWLQ NQRFNGRZQ OLQHV 7ZR ZHHNV
IROORZLQJ LQWUDVSOHQLF LQRFXODWLRQ RI WXPRU FHOOV ZH TXHULHG PLFH IRU KHPDWRJHQRXV
GLVVHPLQDWLRQWRWKHOLYHUDQGVXEVHTXHQWFRORQL]DWLRQ$QDO\VLVRI*)3WXPRUQRGXOHVRQOLYHUV
RI PLFH LQMHFWHG ZLWK WKH 0 FHOO OLQH FRQWDLQLQJ WKH VK51$V UHYHDOHG QR GLIIHUHQFHV LQ
PHWDVWDVLV IRUPDWLRQ EHWZHHQ WKH FRQWURO KDLUSLQ VK/XF DQG WKH JDOHFWLQ KDLUSLQV
6XSSOHPHQWDU\)LJ+VXJJHVWLQJWKDWJDOHFWLQSURGXFWLRQE\WKHPHWDVWDVHVGRHVQRWDIIHFW
FRORQL]DWLRQ7KXVDQ\LQWHUDFWLRQVZLWKJDOHFWLQWKDWSURPRWHPHWDVWDVLVOLNHO\UHVXOWIURPLWV
H[SUHVVLRQE\VWURPDOSRSXODWLRQV
%DVHGRQWKHREVHUYDWLRQWKDWJDOHFWLQGHILFLHQWWXPRUVDUHVWLOOFDSDEOHRILQGXFLQJP\HORLG
FHOO PRELOL]DWLRQ LQWR FLUFXODWLRQ ZH LQYHVWLJDWHG ZKHWKHU RWKHU WXPRUGHULYHG IDFWRUV PLJKW
HOLFLW WKLV PRELOL]DWLRQ :H QH[W DVNHG ZKHWKHU WKH WXPRUV SURGXFH RWKHU F\WRNLQHV RU
FKHPRNLQHV WKDW PD\ UHFUXLW WKH OHXNRF\WHV $QDO\VLV RI WKH &0 E\ /XPLQH[ (/,6$ UHYHDOHG
HOHYDWHGOHYHOVRILQWHUOHXNLQ,/FRPSDUHGWRFRQWUROPHGLXP)LJ&,/LVDSOHRWURSLF
F\WRNLQHNQRZQWRDFWXSRQPXOWLSOHOHXNRF\WHVXEVHWV$VWKH7QRQ0HWOLQHDOVRHOLFLWHGP\HORLG
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
FHOOPRELOL]DWLRQZHH[DPLQHGIl6JHQHH[SUHVVLRQOHYHOVLQDOORIWKHFHOOOLQHVIURPHDFKRIWKH
IRXUJURXSV)LJ'$QDO\VLVRIJHQHH[SUHVVLRQPLFURDUUD\GDWDUHYHDOHGFRQVWDQWH[SUHVVLRQ
RI Il6 DFURVV DOO IRXU FODVVHV RI FHOO OLQHV VXJJHVWLQJ WKDW H[SUHVVLRQ RI ,/ E\ WXPRUV
FRPPHQFHVHDUO\LQWXPRULJHQHVLV
7RGHWHUPLQHWKHUHOHYDQFHRIWXPRUGHULYHG,/WRWKHKXPDQGLVHDVHZHH[DPLQHGJHQRPLF
DPSOLILFDWLRQRIWKHIL6JHQHLQSDWLHQWVZLWKOXQJDGHQRFDUFLQRPDV&RPSDUHGWRQRUPDOEORRG
RUOXQJOXQJDGHQRFDUFLQRPDVKDGVLJQLILFDQWO\HOHYDWHGFRS\QXPEHUVRIWKHIL6JHQH)LJ(
P î 7KXV ZH DVNHG ZKHWKHU ,/ LQGXFHV WKH PRELOL]DWLRQ RI WKH JDOHFWLQ
OHXNRF\WHV LQ RXU V\VWHP ,Q FRQWUDVW WR JDOHFWLQ LQMHFWLRQV RI UHFRPELQDQW ,/ LQGXFHG WKH
UDSLG PRELOL]DWLRQ RI &'EJDOHFWLQ OHXNRF\WHV DQG WKH UHOHYDQW VXESRSXODWLRQV LQWR WKH
FLUFXODWLRQLQDPDQQHUWKDWPLUURUHG&0LQMHFWLRQV)LJ)*DQG6XSSOHPHQWDU\)LJ$'
7R LGHQWLI\ WKH VHW RI OHXNRF\WHV UHVSRQVLEOH IRU JDOHFWLQ SUHVHQWDWLRQ ZH DQDO\]HG WKH
JUDQXORF\WHVDVWKH\PD\UHSUHVHQWDODUJHSRUWLRQRIWKHPRELOL]HG&'E*UFHOOV7KXV
ZH UHSHDWHG WKH ,/ H[SHULPHQWV LQ Csf3r *&6)5 NQRFNRXW PLFH ZKLFK H[KLELW LPSDLUHG
JUDQXORF\WHSURGXFWLRQDQGPDWXUDWLRQ:KLOH,/ZDVVWLOOFDSDEOHRILQGXFLQJ&'E/\&KL
FHOO PRELOL]DWLRQ Csf3r-/- PLFH H[KLELWHG D SURIRXQG UHGXFWLRQ LQ &'EJDOHFWLQ DQG
&'E*U OHXNRF\WHV FRPSDUHG WR ZLOGW\SH PLFH 6XSSOHPHQWDU\ )LJ $' 7KHVH
ILQGLQJV VXJJHVW WKDW WXPRUGHULYHG ,/ LV VXIILFLHQW WR LQGXFH PRELOL]DWLRQ RI JDOHFWLQ
OHXNRF\WHV
7R GHWHUPLQH ZKHWKHU JDOHFWLQ H[SUHVVLRQ E\ WKH P\HORLG FHOOV RU DQRWKHU QRQSDUHQFK\PDO
SRSXODWLRQLVDSUHUHTXLVLWHWRWKHLUPRELOL]DWLRQLQWRFLUFXODWLRQZHUHSHDWHGWKH,/LQMHFWLRQV
LQ Lgals3 JDOHFWLQ NQRFNRXW PLFH DQG REVHUYHG QR GLIIHUHQFHV LQ &'E P\HORLG FHOO
PRELOL]DWLRQFRPSDUHGWRZLOGW\SHPLFH)LJ+DQG6XSSOHPHQWDU\)LJ$(7KHUHOHYDQW
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
VXESRSXODWLRQV DOVR GHPRQVWUDWHG VLPLODU PRELOL]DWLRQ 6XSSOHPHQWDU\ )LJ $' 7DNHQ
WRJHWKHUWKHVHGDWDVXJJHVWWKDW,/DQGQRWJDOHFWLQDFWVV\VWHPLFDOO\DVDIDFWRUWRLQGXFH
WKH PRELOL]DWLRQ RI &'EJDOHFWLQ OHXNRF\WHV IURP WKH ERQH PDUURZ DQG WKDW GLVSOD\ RI
JDOHFWLQE\WKH%0'&VLVQRWDSUHUHTXLVLWHWRWKHLUPRELOL]DWLRQ
,QFUHDVLQJO\PHWDVWDWLFFHOOVH[KLELWHOHYDWHG7$QWLJHQVXUIDFHSUHVHQWDWLRQWKDWPHGLDWHV
JDOHFWLQELQGLQJ
2XU SUHYLRXV VWXGLHV GHPRQVWUDWHG WKDW FHOOV LQFUHDVH WKHLU DGKHVLRQ WR JDOHFWLQ DV WKH\ JDLQ
PHWDVWDWLF SRWHQWLDO ,Q OLJKW RI WKH HQULFKPHQW RI JDOHFWLQ OHXNRF\WHV LQ WKH SHULSKHUDO
EORRG)LJDQGPHWDVWDWLFQLFKH)LJ'ZHDVNHGKRZWKHPHWDVWDWLFSRSXODWLRQVDFKLHYH
HQKDQFHG DGKHVLRQ WR JDOHFWLQ $V JDOHFWLQV ELQG ȕJDODFWRVLGH JO\FDQV WKURXJK WKHLU
FDUERK\GUDWH UHFRJQLWLRQ GRPDLQ &5' ZH DVNHG ZKHWKHU FKDQJHV LQ JO\FDQ SUHVHQWDWLRQ E\
WXPRU FHOOV FRQIHU WKHLU DGKHVLRQ WR JDOHFWLQ RQ UHFUXLWHG OHXNRF\WHV GXULQJ PHWDVWDVLV
$QDO\VLV RI JO\FDQ PLFURDUUD\ GDWDVHWV DQG H[LVWLQJ OLWHUDWXUH UHYHDO D YDULHW\ RI
FDUERK\GUDWH OLJDQGV IRU WKHVH JDOHFWLQV )LJ $ :KLOH DOO JDOHFWLQV ELQG ODFWRVDPLQHV
JDOHFWLQH[KLELWVVSHFLILFDIILQLWLHVIRUWKH7KRPVHQ)ULHGHQUHLFK$QWLJHQ7$QWLJHQ*DOȕ
*DO1$FĮ6HU7KUDSDQFDUFLQRPDPDUNHUH[SUHVVHGRQPDQ\JO\FRSURWHLQV)LJ%
*DOHFWLQ LV XQDEOH WR LQWHUDFW ZLWK WKH 7$QWLJHQ LI WKH GLVDFFKDULGH LV IXUWKHU JO\FRV\ODWHG
7KXVZHDVNHGZKHWKHUPHWDVWDWLFFHOOVH[KLELWHOHYDWHG7$QWLJHQOHYHOVRQWKHLUVXUIDFHVWKDW
PLJKW PHGLDWH LQWHUDFWLRQV ZLWK JDOHFWLQ 6WDLQLQJ RI 7QRQ0HW 70HW DQG 0 OLQHV ZLWK D 7
$QWLJHQVSHFLILF OHFWLQSHDQXW DJJOXWLQLQ 31$ UHYHDOHG WKDW WKH OHYHOV RI 7$QWLJHQ VXUIDFH
SUHVHQWDWLRQaIROGLQFUHDVHFRUUHODWHZLWKPHWDVWDWLFSRWHQWLDO)LJ&DQG6XSSOHPHQWDU\
)LJ $ 7R GHWHUPLQH ZKHWKHU PHWDVWDWLF FHOO DGKHVLRQ WR JDOHFWLQ LV LQGHHG FDUERK\GUDWH
PHGLDWHG ZH LQFXEDWHG WKH FHOOV ZLWK IOXRUHVFHQW JDOHFWLQ LQ WKH SUHVHQFH RI D FRPSHWLWLYH
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
ELQGHU N$FHW\OODFWRVDPLQH /DF1$F )ORZ F\WRPHWU\ UHYHDOHG D UHGXFWLRQ LQ ELQGLQJ LQ WKH
SUHVHQFH RI /DF1$F FRPSDUHG WR FRQWURO VXFURVH VXJJHVWLQJ WKDW JDOHFWLQ DGKHVLRQ WR WKH
PHWDVWDWLFFHOOVLVFDUERK\GUDWHPHGLDWHG)LJ'
7R LQYHVWLJDWH ZKHWKHU WKHVH DOWHUDWLRQV DUH REVHUYHG LQ WKH KXPDQ GLVHDVH ZH VWDLQHG KXPDQ
16&/& WLVVXH PLFURDUUD\V RI WLVVXH VDPSOHV ZLWK 31$ WR DVVHVV WKH SUHVHQFH RI WKH 7
$QWLJHQ 7KLV VWDLQLQJ UHYHDOHG HOHYDWHG OHYHOV RI FHOOVXUIDFH 7$QWLJHQ SUHVHQWDWLRQ LQ
FDQFHURXVWLVVXHFRPSDUHGWRQRQFDQFHURXVWLVVXHZLWKDQRWDEOHHQULFKPHQWLQWKHO\PSKQRGH
PHWDVWDVHV )LJ () DQG 6XSSOHPHQWDU\ )LJ % $GGLWLRQDOO\ ZH H[DPLQHG IRXU FRPPRQ
16&/&FHOOOLQHVIRUWKHLUUHODWLYHH[SUHVVLRQRIWKH7$QWLJHQ)ORZF\WRPHWU\UHYHDOHGWKDW
LQGHHG WKUHH RI WKH IRXU OLQHV ERXQG 31$ DW FRPSDUDEOH OHYHOV WR WKH 0 OLQH ZLWK WKH PRVW
LQYDVLYH RI WKH OLQHV $ H[KLELWLQJ WKH KLJKHVW GHJUHH RI VWDLQLQJ )LJ * 7DNHQ
WRJHWKHUWKHVHGDWDVXJJHVWWKDWOXQJWXPRUVELQGJDOHFWLQWKURXJKFDUERK\GUDWHHSLWRSHVVXFK
DVWKH7$QWLJHQDQGWKDWWKLVHSLWRSHLVH[SUHVVHGPRUHKLJKO\RQPHWDVWDWLFFHOOV
'LIIHUHQWLDO J\FRV\OWUDQVIHUDVH H[SUHVVLRQ SURPRWHV LQFUHDVHG 7$QWLJHQ SUHVHQWDWLRQ
WKURXJKUHGXFWLRQRIJO\FDQFKDLQHORQJDWLRQDQGLQFUHDVHGFDSSLQJ
$ YDULHW\ RI OOLQNHG JO\FRSURWHLQV VXFK DV 08& FDQ H[KLELW SURIRXQG GHJUHHV RI O
JO\FRV\ODWLRQDQGGLIIHUHQWLDOUHJXODWLRQRIVXFKJO\FRSURWHLQVFDQVLJQLILFDQWO\DOWHUWKHRYHUDOO
JO\FDQSUHVHQWDWLRQRIFHOOV7KXVZHDVNHGZKHWKHULQFUHDVHG7$QWLJHQH[SUHVVLRQLVDUHVXOW
RI XSUHJXODWLRQ RI LQGLYLGXDO JO\FRSURWHLQV RU DOWHUHG JO\FRV\OWUDQVIHUDVH DFWLYLW\ :H LVRODWHG
FHOOPHPEUDQHSURWHLQVIURP7QRQ0HW70HWDQG0OLQHVDQGSHUIRUPHG31$OHFWLQEORWV7KHVH
UHYHDOHGDJOREDOLQFUHDVHLQ7$QWLJHQH[SUHVVLRQDFURVVPDQ\JO\FRSURWHLQVLQWKHLQFUHDVLQJO\
PHWDVWDWLFFHOOVDVRSSRVHGWR31$ODEHOLQJRIDVSHFLILFJO\FRSURWHLQ)LJ$
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
,QOLJKWRIWKLVEURDGGLVWULEXWLRQLQJO\FRV\ODWLRQZHLQYHVWLJDWHGDOWHUDWLRQVLQWKHH[SUHVVLRQ
DQG DFWLYLW\ RI JO\FRV\OWUDQVIHUDVHV $QDO\VLV RI JHQH H[SUHVVLRQ PLFURDUUD\V IRU JO\FRV\OWUDQVIHUDVH JHQHV GLG QRW GHPRQVWUDWH ZLGHVSUHDG VKLIWV LQ H[SUHVVLRQ QRU ZHUH WKHUH
VLJQLILFDQWFKDQJHVLQWKHVXEVHWRIJHQHVWKDWHQFRGHWUDQVIHUDVHVVSHFLILFIRUJHQHUDWLRQRIWKH
JDOHFWLQ OLJDQGV 6XSSOHPHQWDU\ )LJ $ % &RPSDULVRQV EHWZHHQ WKH DYHUDJHV IRU WKH
SULPDU\ WXPRU DQG PHWDVWDVLVGHULYHG FHOO OLQHV KRZHYHU LGHQWLILHG Gcnt3 DQG St6galnac4 DV
EHLQJXQGHUDQGRYHUH[SUHVVHGLQWKHPHWDVWDWLFOLQHVUHVSHFWLYHO\)LJ%WKHVHDOWHUDWLRQV
ZHUH FRQILUPHG E\ T573&5 LQ WKH UHSUHVHQWDWLYH FHOO OLQHV )LJ & WRS &*Q7 Gcnt3
LQGXFHVWKHDGGLWLRQRIDȕ*OF1$FWRWKH7$QWLJHQZKHUHDV6W*DO1$F,9DGGVDQĮ
1HX$F WR WKH VLDO\O7$QWLJHQ )LJ & ERWWRP $V IXUWKHU HORQJDWLRQ RI WKH FRUH GLVDFFKDULGHSUHYHQWVELQGLQJE\JDOHFWLQ)LJ%FKDQJHVLQWUDQVIHUDVHDFWLYLW\FRQVLVWHQW
ZLWK WKH H[SUHVVLRQ FKDQJHV LQ Gcnt3 DQG St6galnac4 ZRXOG SURPRWH 7$QWLJHQ SUHVHQWDWLRQ
WKURXJK SUHYHQWLRQ RI EUDQFKLQJ DQG FDSSLQJ RI HORQJDWLRQ UHVSHFWLYHO\ *HQH H[SUHVVLRQ
PLFURDUUD\ DQDO\VLV RI WKH RWKHU FHOO OLQHV RI HDFK FODVV UHYHDOHG VLPLODU WUHQGV IRU WKHVH DQG
DQDORJRXVWUDQVIHUDVHV6XSSOHPHQWDU\)LJ&RQVLVWHQWZLWKWKHIDFWWKDWJHQHUDWLRQRI WKH
7$QWLJHQRFFXUVLQWKH*ROJLSULRUWRVXEVHTXHQWJO\FRV\ODWLRQQRGLIIHUHQFHLQ7$QWLJHQ
EORWWLQJZDVYLVLEOHEHWZHHQZKROHFHOOO\VDWHVRI70HWDQG0OLQHV6XSSOHPHQWDU\)LJ$
IXUWKHU FRQILUPLQJ WKDW GLIIHUHQFHV LQ LWV SUHVHQWDWLRQ DUH GXH WR GHFUHDVHG JO\FDQ H[WHQVLRQ
UDWKHUWKDQLQFUHDVHGSURGXFWLRQRIWKHGLVDFFKDULGH
7R GHWHUPLQH WKH UHOHYDQFH RI WKH FDSSLQJ DQG EUDQFKLQJ WUDQVIHUDVHV WR KXPDQ GLVHDVH ZH
DJDLQH[DPLQHGFRS\QXPEHURIWKHGCNT3JHQHLQWKHKXPDQ7&*$OXQJFDQFHUGDWDVHWV7KLV
DQDO\VLVUHYHDOHGDGHFUHDVHLQFRS\QXPEHULQOXQJDGHQRFDUFLQRPDFRPSDUHGWRQRUPDOEORRG
RUOXQJ)LJ'P î$GGLWLRQDOO\ZHH[DPLQHGJHQHH[SUHVVLRQRIWKHUHOHYDQW
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
JO\FRV\OWUDQVIHUDVHJHQHVLQWKHIRXUKXPDQ16&/&FHOOOLQHVWKDWH[KLELWHGYDU\LQJGHJUHHVRI
31$VWDLQLQJ:KLOHPLQLPDOGLIIHUHQFHVLQH[SUHVVLRQZHUHREVHUYHGIRUWKHWUDQVIHUDVHVWKDW
JLYH ULVH WR WKH FRUH GLVDFFKDULGH C1GALT1 DQG C1GALT1C1 WKHUH ZHUH PDUNHG YDULDQFHV
DFURVV WKH OLQHV IRU WKH JHQHV HQFRGLQJ WUDQVIHUDVHV WKDW SURGXFH WKH EUDQFKHG DQG FDSSHG
VWUXFWXUHV 6XSSOHPHQWDU\ )LJ % 7KHVH GDWD VXJJHVW WKDW DOWHUDWLRQV LQ JO\FRV\OWUDQVIHUDVH
DFWLYLW\ WKDW UHJXODWH FDSSLQJ RU EUDQFKLQJ RI WKH 7$QWLJHQ PD\ DOVR SHUWDLQ WR WKH KXPDQ
GLVHDVH
5HJXODWLRQRIGcnt3DQGSt6galnac4PHGLDWHJDOHFWLQDGKHVLRQin vitroDQGPHWDVWDVLVin
vivo
7R WHVW WKH IXQFWLRQDO UROH RI WKHVH WUDQVIHUDVHV RQ JDOHFWLQ ELQGLQJ DQG PHWDVWDVLV ZH
WUDQVIHFWHG WKH PHWDVWDWLF FHOOV ZLWK Gcnt3 RU NQRFNHG GRZQ St6galnac4 ZLWK VKRUW KDLUSLQV
7UDQVIHFWLRQ RI Gcnt3 UHVXOWHG LQ GHFUHDVHG 7$QWLJHQ SUHVHQWDWLRQ DQG GHFUHDVHG JDOHFWLQ
ELQGLQJ DQG LQ D FRPSOHPHQWDU\ IDVKLRQ NQRFNGRZQ RI St6galnac4 \LHOGHG D UHGXFWLRQ LQ
JDOHFWLQ ELQGLQJ ZKLOH KDYLQJ QR HIIHFW RQ QRQVLDO\ODWHG 7$QWLJHQ SUHVHQWDWLRQ DV 31$
GRHV QRW UHFRJQL]H VLDO\ODWHG IRUPV RI WKH DQWLJHQ )LJ $ DQG 6XSSOHPHQWDU\ )LJ $(
$ % 7R GHWHUPLQH WKH IXQFWLRQDO UROH RI 6W*DO1$F,9 DFWLYLW\ RQ PHWDVWDVLV in vivo ZH
SHUIRUPHGH[SHULPHQWDOPHWDVWDVLVDVVD\VZLWKWKHSt6galnac4NQRFNGRZQOLQH&HOOVH[SUHVVLQJ
DKDLUSLQIRUWKHVLDO\OWUDQVIHUDVHRUDFRQWUROKDLUSLQDJDLQVWILUHIO\OXFLIHUDVHZHUHLQMHFWHGLQWR
WKH VSOHHQV RI PLFH DQG PRQLWRUHG IRU OLYHU WXPRU IRUPDWLRQ IROORZLQJ KHPDWRJHQRXV
GLVVHPLQDWLRQ:HIRXQGWKDWPLFHLQMHFWHGZLWKWKH0VKSt6galnac4FHOOVH[KLELWHGJUHDWHU
WKDQ IHZHU OLYHU PHWDVWDVHV WKDQ FRQWURO PLFH WZR ZHHNV IROORZLQJ WXPRU FHOO LQMHFWLRQ
)LJ %& DQG 6XSSOHPHQWDU\ )LJ :H REVHUYHG QR GLIIHUHQFHV LQ SUROLIHUDWLRQ UDWHV
EHWZHHQ WKH NQRFNGRZQ DQG FRQWURO KDLUSLQ FHOO OLQHV in vitro 6XSSOHPHQWDU\ )LJ 7DNHQ
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
WRJHWKHU WKHVH GDWD VXJJHVW WKDW DOWHUDWLRQV LQ JO\FRV\OWUDQVIHUDVH DFWLYLW\ REVHUYHG LQ
LQFUHDVLQJO\ PHWDVWDWLF FHOOV KDYH D IXQFWLRQDO LPSDFW RQ WKH PHWDVWDWLF SRWHQWLDO RI WKH WXPRU
FHOOV DQG VXSSRUW WKH K\SRWKHVLV WKDW SUHVHQWDWLRQ RI WKH 7$QWLJHQ WKURXJK WKH SUHYHQWLRQ RI
IXUWKHUJO\FRV\ODWLRQSURPRWHVDGKHVLRQRIWXPRUFHOOVWRJDOHFWLQLQWKHPHWDVWDWLFQLFKH
'LVFXVVLRQ
$ YDULHW\ RI PROHFXOHV KDYH EHHQ GHPRQVWUDWHG WR SOD\ LPSRUWDQW UROHV LQ FRORQL]DWLRQ RI WKH
HDUO\PHWDVWDWLFQLFKHWKURXJKWKHLUV\VWHPLFVHFUHWLRQDQGE\LQWHUDFWLQJZLWKUHFUXLWHG&'E
OHXNRF\WHV7KHLGHQWLILFDWLRQRIGLUHFWLQWHUDFWLRQVEHWZHHQWKHVHOHXNRF\WHVDQGWXPRU
FHOOV ZLWKLQ WKH EORRG YHVVHOV DQG FRORQL]HG WLVVXHV VXJJHVWV D UROH IRU D FRQVHUYHG DGKHVLRQ
UHFHSWRUOLJDQG D[LV GXULQJ PHWDVWDWLF SURJUHVVLRQ +HUH ZH LGHQWLI\ JDOHFWLQ DV D QRYHO
UHJXODWRURIOXQJDGHQRFDUFLQRPDPHWDVWDVLVWKURXJKLWVSUHVHQWDWLRQZLWKLQWKHPHWDVWDWLFQLFKH
DQG ELQGLQJ WR WKH RQFRIHWDO GLVDFFKDULGH NQRZQ DV WKH 7$QWLJHQ )LJ %DVHG RQ RXU
ILQGLQJV ZH SURSRVH WKDW HDUO\ LQ WXPRULJHQHVLV WXPRUV VHFUHWH VROXEOH IDFWRUV VXFK DV ,/
WKDW LQGXFH WKH PRELOL]DWLRQ RI &'EJDOHFWLQ %0'&V LQWR FLUFXODWLRQ $GGLWLRQDOO\
JDOHFWLQLVSUHVHQWHGRQPDFURSKDJHVLQWKHHDUO\PHWDVWDWLFQLFKHRIWXPRUEHDULQJPLFH$V
WXPRUV JDLQ PHWDVWDWLF SRWHQWLDO WKH\ LQFUHDVH WKHLU SUHVHQWDWLRQ RI WKH 7$QWLJHQ RQ WKHLU
VXUIDFHV ,Q WKH FRQWH[W RI RXU OXQJ DGHQRFDUFLQRPD PRXVH PRGHO ZH VKRZ WKDW WKLV HOHYDWHG
SUHVHQWDWLRQGRHVQRWUHVXOWIURPLQFUHDVHGOJO\FRV\ODWLRQWKURXJKFRUHJO\FRV\OWUDQVIHUDVHV
EXW UDWKHU LV GXH WR WKH DEURJDWLRQ RI FRUH FKDLQ H[WHQVLRQ FRQIHUUHG E\ GRZQUHJXODWLRQ RI
&*Q7DQGFDSSLQJRIWKHPRWLIE\WKHĮVLDO\OWUDQVIHUDVH6W*DO1$F,97KHVHFKDQJHVLQ
WUDQVIHUDVH DFWLYLW\ DQG 7$QWLJHQ SUHVHQWDWLRQ UHVXOW LQ LQFUHDVHG FDUERK\GUDWHGHSHQGHQW
ELQGLQJ RI WKH WXPRU FHOOV WR JDOHFWLQ DQG LQFUHDVHG PHWDVWDVLV in vivo $V WKH SUHVHQFH RI
WXPRUV LQGXFHV HOHYDWHG QXPEHUV RI JDOHFWLQ P\HORLG FHOOV LQ WKH FLUFXODWLRQ DQG PHWDVWDWLF
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
QLFKHWKHVHDOWHUDWLRQVLQWUDQVIHUDVHDFWLYLW\ZLWKLQWKHWXPRUFHOOVPD\UHSUHVHQWDQHVVHQWLDO
PHFKDQLVP E\ ZKLFK PHWDVWDWLF FHOOV LQWHUDFW ZLWK WKHVH SURWXPRULJHQLF OHXNRF\WHV DW WKRVH
VLWHV)XUWKHUPRUHSHUWXUEDWLRQVLQH[SUHVVLRQRIWKHVHWUDQVIHUDVHVDUHFRUUHODWHGZLWKKXPDQV
ZLWK 16&/& )LJ DQG 6XSSOHPHQWDU\ )LJ % DQG WKH 7$QWLJHQ LV IUHTXHQWO\
RYHUH[SUHVVHGRQPDQ\FDQFHUVVXJJHVWLQJWKDWWKLVPRGHORIJO\FDQUHJXODWLRQPD\H[WHQG
WRPDQ\FOLQLFDOVHWWLQJV
'LVVHPLQDWLRQ RI WXPRU FHOOV LV WKRXJKW WR RFFXU HDUO\ LQ WXPRULJHQHVLV 5HFHQW HYLGHQFH
VXSSRUWVWKHFRQFHSWWKDWWKHUDWHOLPLWLQJVWHSLQWKHIRUPDWLRQRIFOLQLFDOO\UHOHYDQWWXPRUVLV
WKH DGDSWDWLRQ RI GLVVHPLQDWHG FDQFHU FHOOV WR WKHLU ORFDO PLFURHQYLURQPHQWV LQ D PDQQHU WKDW
SHUPLWV FRORQL]DWLRQ $GGLWLRQDO VWXGLHV KDYH VXJJHVWHG WKDW HDUO\ RU HYHQ ³SUH´
PHWDVWDWLFQLFKHVPD\H[LVWZKHUHLQDSURWXPRULJHQLFPLFURHQYLURQPHQWLVJHQHUDWHG6XFK
PLFURHQYLURQPHQWVKDUERULQIODPPDWRU\OHXNRF\WHVWKDWDFWWRSURPRWHPHWDVWDWLFFRORQL]DWLRQ
2XUILQGLQJVRQWKHPRELOL]DWLRQDQGUHFUXLWPHQWRIWKHVHLQIODPPDWRU\SRSXODWLRQV
E\ QRQPHWDVWDWLF SULPDU\ WXPRUV DUH FRQVLVWHQW ZLWK WKH K\SRWKHVLV WKDW LQYROYHPHQW RI SUR
PHWDVWDWLF VWURPDO SRSXODWLRQV LV DW OHDVW LQSDUW DQ HDUO\ HYHQW 7KXV DGDSWDWLRQV E\ WKH
PHWDVWDWLFWXPRUFHOOVWKDWSRWHQWLDWHLQWHUDFWLRQVZLWKWKHVHOHXNRF\WHVPD\UHSUHVHQWWKHUDWH
OLPLWLQJHYHQWVSHUPLWWLQJFRORQL]DWLRQ+HUHZHILQGWKDWDOWHUDWLRQVLQJO\FRV\ODWLRQWKDWUHVXOW
LQ 7$QWLJHQ SUHVHQWDWLRQ LV RQH VXFK HYHQW LQ WKDW LW RFFXUV ODWHU LQ WXPRU SURJUHVVLRQ DQG
SURPRWHVLQWHUDFWLRQVZLWKJDOHFWLQLQWKHPHWDVWDWLFQLFKH
$ YDULHW\ RI VWXGLHV KDYH LPSOLFDWHG JDOHFWLQ LQ WXPRU SURJUHVVLRQ DQG PHWDVWDVLV 7KH
PDMRULW\ RI WKHVH VWXGLHV KDYH GHPRQVWUDWHG VWURQJ FOLQLFDO FRUUHODWLRQV ZLWK DGYDQFHG VWDJH
GLVHDVHRUGHFUHDVHGVXUYLYDOZLWKHOHYDWHGOHYHOVRIJDOHFWLQ,QDJUHHPHQWZLWKWKHVH
FOLQLFDOUHSRUWVRXUPRGHODOVRGHPRQVWUDWHGHOHYDWHGVHUXPOHYHOVRIJDOHFWLQLQPLFHEHDULQJ
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
WXPRUV 6XSSOHPHQWDU\ )LJ ([SHULPHQWDO VWXGLHV KDYH SULPDULO\ LQYHVWLJDWHG WKH UROHV RI
JDOHFWLQVLQPRGXODWLQJWKHLPPXQHV\VWHPRULQLQWHUDFWLRQVZLWKEORRGYHVVHOV,QDGGLWLRQ
WR LWV UHSRUWHG UROH LQ SURPRWLQJ DQJLRJHQHVLV VXUIDFH H[SUHVVLRQ RI JDOHFWLQ RQ
HQGRWKHOLDOFHOOVKDVEHHQVXJJHVWHGWRSURPRWHDGKHVLRQRIEUHDVWDQGSURVWDWHFDQFHUFHOOVWR
WKH HQGRWKHOLXP WKURXJK FDUERK\GUDWH LQWHUDFWLRQV +HUH ZH VXJJHVW DQ DGGLWLRQDO UROH IRU
JDOHFWLQ LQ PHWDVWDVLV LQ WKDW LW LV SUHVHQWHG ZLWKLQ WKH HDUO\ PHWDVWDWLF QLFKH RQ UHFUXLWHG
PHWDVWDVLVDVVRFLDWHG P\HORLG FHOOV 7KXV JDOHFWLQ PD\ QRW DFW RQO\ WR SURPRWH DGKHVLRQ WR
HQGRWKHOLXP EXW DOVR WR SURPRWH LQWHUDFWLRQV EHWZHHQ WKH FDQFHUFHOOVDQG %0'&V ZLWKLQWKH
WLVVXHV RU ZLWKLQ FLUFXODWLRQ LQ D PDQQHU WKDW QRW RQO\ SURPRWHV H[WUDYDVDWLRQ EXW DOVR
FRORQL]DWLRQ
$QDEXQGDQFHRIFOLQLFDODQGH[SHULPHQWDOHYLGHQFHKDVVXJJHVWHGDUROHIRULQWHUOHXNLQ,/
LQFDQFHUPHWDVWDVLV7XPRUGHULYHGVROXEOHIDFWRUVLQFOXGLQJDYDULHW\RI(&0
PROHFXOHV VXFK DV ODPLQLQV DQG YHUVLFDQ KDYH EHHQ UHSRUWHG WR DFW XSRQ PDFURSKDJHV
WKURXJK 7ROOOLNH UHFHSWRUV 7/5V WR LQGXFH WKH SURGXFWLRQ RI ,/ +HUH ZH ILQG WKDW OXQJ
WXPRUV WKHPVHOYHV SURGXFH ,/ LQGHSHQGHQW RI WKH SUHVHQFH RI WKH P\HORLG FHOOV 2XU
FRQGLWLRQHG PHGLXP H[SHULPHQWV VXJJHVW WKDW VROXEOH IDFWRUV VXFK DV ,/ DUH FDSDEOH RI
LQGXFLQJ WKH UDSLG PRELOL]DWLRQ RI &'E/\&KL DQG &'E*U P\HORLG FHOOV LQWR
FLUFXODWLRQZKLFKDUHWKRXJKWWRSURPRWHPHWDVWDVLV2WKHUPROHFXOHVWUDGLWLRQDOO\FRQVLGHUHGWR
EH SURGXFHG E\ P\HORLG FHOOV VXFK DV &&/ FDQ EH SURGXFHG E\ WXPRU FHOOV WR LQGXFH WKH
UHFUXLWPHQWRISURPHWDVWDWLF&'ESRSXODWLRQV7KHSURGXFWLRQRIF\WRNLQHVVXFKDV,/
E\WXPRUVUHSUHVHQWVDQRWKHUPHDQVE\ZKLFKWXPRUVFDQLQGLUHFWO\SURPRWHWKHGHYHORSPHQW
RILQIODPPDWRU\PLFURHQYLURQPHQWVDWGLVWDQWVLWHV
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
$OWHUDWLRQVLQJO\FRV\ODWLRQKDYHORQJEHHQDVVRFLDWHGZLWKPDOLJQDQF\0DQ\FOLQLFDO
ELRPDUNHUV DUH FDUERK\GUDWH DQWLJHQV VXFK DV &$ &$ '83$1,, DQG $)3/ )XUWKHUPRUH RYHUH[SUHVVLRQ RI PDQ\ OOLQNHG JO\FRSURWHLQV VXFK DV 08& KDYH EHHQ
FRUUHODWHGZLWKPDOLJQDQF\$VDUHVXOWWKLVJO\FRSURWHLQKDVEHHQWKHVXEMHFWRIQXPHURXV
YDFFLQH GHYHORSPHQW LQLWLDWLYHV DQG WKHVH YDFFLQHV DUH W\SLFDOO\ PRUH HIILFDFLRXV ZKHQ WKH
JO\FRV\ODWHG DQWLJHQ LV XVHG ,Q DGGLWLRQ WR H[SUHVVLRQ RI SDUWLFXODU JO\FRSURWHLQV
DEHUUDQWJO\FRV\OWUDQVIHUDVHDFWLYLW\LVIUHTXHQWO\REVHUYHGLQDYDULHW\RIFDQFHUV:HVKRZWKDW
XSUHJXODWLRQ RI 6W*DO1$F,9 DQG GRZQUHJXODWLRQ &*Q7 SUHVHUYH SUHVHQWDWLRQ RI WKH 7
$QWLJHQ WKURXJK SUHYHQWLRQ RI OJO\FDQ FKDLQ HORQJDWLRQ 7RJHWKHU ZLWK SUHYLRXV ILQGLQJV RI
RYHUH[SUHVVLRQ RI ST6GALNAC5 LQ EUHDVW FDQFHU PHWDVWDVLV WR WKH EUDLQ DQG JHQHUDO
LQFUHDVHVLQVLDO\ODWLRQRQ PHWDVWDWLFFHOOOLQHVRXUVWXG\VXJJHVWVDFRQVHUYHGPHFKDQLVP
E\ ZKLFK WKH 7$QWLJHQ LV SUHVHQWHG RQ PHWDVWDVL]LQJ FHOOV WKDW DSSHDUV WR SRWHQWLDWH
FRORQL]DWLRQWKURXJKLQWHUDFWLRQVZLWKJDOHFWLQVLQWKHPHWDVWDWLFQLFKH,WLVZRUWKQRWLQJWKDWD
UHFHQW VWXG\ IRXQG WKDW DQRWKHU VLDO\OWUDQVIHUDVH 67*DO1$F,, PD\ LQIOXHQFH PHWDVWDWLF
SRWHQWLDO,QWKLVFDVHKRZHYHUNQRFNGRZQRI67*DO1$F,,LQDEUHDVWFDQFHUFHOOOLQHOHG
WR DQ LQFUHDVH LQ PHWDVWDVLV :KLOH RQ WKH VXUIDFH WKLV ILQGLQJ DSSHDUV WR FRQWUDGLFW WKRVH
SUHVHQWHG ZLWKLQ WKLV PDQXVFULSW WKH GDWD DUH FRQVLVWHQW LQ WKDW LQ ERWK FDVHVJO\FRV\ODWLRQ
GHSHQGHQWJDOHFWLQELQGLQJSURPRWHVPHWDVWDVLVIRUPDWLRQ2QHSRVVLELOLW\LVWKDWWKHLQYHUVH
UHJXODWLRQ RI WKH WZR WUDQVIHUDVHV PD\ UHVXOW LQ WKH VDPH IXQFWLRQDO SKHQRW\SH ZLWK
67*DO1$F,, SUHYHQWLQJ SUHVHQWDWLRQ RI WKH JDOHFWLQ OLJDQG 1$FHW\OODFWRVDPLQH RU VLPLODU
OLJDQGV DQG 67*DO1$F,9 SURPRWLQJ H[SUHVVLRQ RI WKH 7$QWLJHQ 3UHYLRXVO\ ZH IRXQG WKDW
WKHLQWHJULQĮȕSURPRWHVPHWDVWDVLVWKURXJKELQGLQJWRFRPELQDWLRQVRIILEURQHFWLQZLWKHLWKHU
JDOHFWLQ RU ,QWHJULQV FRQWDLQ D YDULHW\ RI JO\FRV\ODWLRQ VLWHV DQG WKHVH GDWD PD\
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
VXJJHVWWKDWJO\FRV\ODWLRQRILQWHJULQVFRXOGSURPRWHPHWDVWDVLVWKURXJKV\QHUJLVWLFLQWHUDFWLRQV
EHWZHHQ JDOHFWLQV DQG RWKHU (&0 PROHFXOHV ZLWKLQ WKH PHWDVWDWLF QLFKH 5HJDUGOHVV RI WKH
SDUWLFXODU SURWHLQV H[KLELWLQJ WKHVH JO\FRV\ODWLRQ SDWWHUQV RXU REVHUYDWLRQV RI DOWHUDWLRQV LQ
WUDQVIHUDVHH[SUHVVLRQDQG7$QWLJHQSUHVHQWDWLRQLQKXPDQVZLWKWKHGLVHDVHVXJJHVWVDUROHIRU
D KLJKO\ FRQVHUYHG PHFKDQLVP RI JO\FDQPHGLDWHG OXQJ FDQFHU PHWDVWDVLV WKDW ZDUUDQWV
FRQVLGHUDWLRQIRUWKHUDSHXWLFLQWHUYHQWLRQ
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
0HWKRGV
&HOO/LQHVDQG0RXVH7UDQVSODQWDWLRQ$VVD\V
0XULQH FHOO OLQHV D JLIW IURP 7\OHU -DFNV DQG 0RQWH :LQVORZ 0DVVDFKXVHWWV ,QVWLWXWH RI
7HFKQRORJ\&DPEULGJH0$ZHUHGHVFULEHGSUHYLRXVO\DQGZHUHFXOWXUHGLQ'0(0ZLWK
YROYRO IHWDO ERYLQH VHUXP DQG /JOXWDPLQH 7KH FHOO OLQHV ZHUH XVHG ZLWKLQ ILYH
SDVVDJHVRIWKHGLVWULEXWHGYLDOVDQGZHUHWHVWHGIUHTXHQWO\IRUFRQVLVWHQF\LQJHQHH[SUHVVLRQE\
T573&51RDGGLWLRQDODXWKHQWLFDWLRQZDVSHUIRUPHG$OOFHOOOLQHVZHUHWHVWHGIRUSDWKRJHQV
E\ 3&5 E\ WKH 0,7 'LYLVLRQ RI &RPSDUDWLYH 0HGLFLQH GXULQJ WKH WLPH SHULRG LQ ZKLFK WKH\
ZHUHEHLQJXVHG7KHKXPDQ16&/&OLQHVZHUHSURYLGHGE\WKH1DWLRQDO&DQFHU,QVWLWXWH1&,
WKURXJK WKH 'LYLVLRQ RI &DQFHU 7UHDWPHQW DQG 'LDJQRVLV '&7' 7XPRU 5HSRVLWRU\ 7KH
UHSRVLWRU\ DXWKHQWLFDWHV WKH OLQHV DQG WHVWV DOO FHOO OLQHV IRU SDWKRJHQV E\ 3&5 SULRU WR
GLVWULEXWLRQ([SHULPHQWVZHUHSHUIRUPHGZLWKLQILYHSDVVDJHVRIWKHGLVWULEXWHGYLDOV
$OO DQLPDO SURFHGXUHV ZHUH SHUIRUPHG LQ DFFRUGDQFH ZLWK WKH 0,7 ,QVWLWXWLRQDO $QLPDO &DUH
DQG 8VH &RPPLWWHH XQGHU SURWRFRO &HOO LQMHFWLRQ VWXGLHV ZHUH SHUIRUPHG LQ
IHPDOH %6)- PLFH ZHHNV RI DJH -DFNVRQ /DERUDWRU\ 6WRFN 1XPEHU $GGLWLRQDOO\ ,/ H[SHULPHQWV ZHUH SHUIRUPHG LQ %;&J&VIUWP/LQN- DQG %&J
/JDOVWP3RL- PLFH -DFNVRQ /DERUDWRU\ 6WRFN 1XPEHUV DQG ZLWK &%/-
PLFH DV FRQWUROV -DFNVRQ /DERUDWRU\ 6WRFN 1XPEHU )RU &'E PRELOL]DWLRQ
H[SHULPHQWV PLFH ZHUH LQMHFWHG ZLWK î FHOOV UHVXVSHQGHG LQ —/ RI 3%6 LQWR WKH
VXEFXWDQHRXV UHJLRQ RI IODQNV RI PLFH ZKLOH DQHVWKHWL]HG ZLWK LVRIOXRUDQH 7XPRUV DQG
SHULSKHUDO EORRG ZHUH KDUYHVWHG ZHHNV IROORZLQJ LQMHFWLRQV $SSUR[LPDWHO\ P/ RI
SHULSKHUDO EORRG ZDV KDUYHVWHG E\ FDUGLDF SXQFWXUH ZKLOH PLFH ZHUH DQHVWKHWL]HG ZLWK
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
LVRIOXRUDQH DQG XVHG IRU DQDO\VLV E\ IORZ F\WRPHWU\ 0LFH ZHUH LPPHGLDWHO\ HXWKDQL]HG
IROORZLQJUHFRYHU\RIWKHEORRGDQGWKHLUWXPRUVZHUHKDUYHVWHG
,QWUDVSOHQLF WUDQVSODQWDWLRQ DVVD\V ZHUH SHUIRUPHG DV SUHYLRXVO\ GHVFULEHG %ULHIO\ î
FHOOVZHUHUHVXVSHQGHGLQ—/RISKRVSKDWHEXIIHUHGVDOLQH3%6$QLPDOVZHUHDQHVWKHWL]HG
ZLWK LVRIOXRUDQH DQG DGPLQLVWHUHG EXSUHQRUSKLQH —JNJ )XU ZDV UHPRYHG ZLWK
VXUJLFDOFOLSSHUVDQGWKHLQFLVLRQVLWHZDVVWHULOL]HGZLWK%HWDGLQHDQGHWKDQRO7KHVSOHHQ
ZDVH[SRVHGWKURXJKDVPDOOLQFLVLRQDQGFHOOVZHUHLQMHFWHGLQWRWKHWLSRIWKHVSOHHQZLWKD
JDXJH QHHGOH 7KH FHOOV ZHUH DOORZHG WR WUDYHO WKURXJK FLUFXODWLRQ IRU WZR PLQXWHV SULRU WR
UHPRYDO RI WKH VSOHHQ 7KH VSOHQLF YHVVHOV ZHUH WKHQ FDXWHUL]HG DQG WKH HQWLUH VSOHHQ ZDV
UHPRYHG7KHPXVFOHZDOOZDVFORVHGZLWKGLVVROYDEOHVXWXUHVDQGWKHVNLQZDVFORVHGZLWK
PPZRXQGFOLSV5RER]0LFHZHUHHXWKDQL]HGWZRZHHNVIROORZLQJLQMHFWLRQDQGWKHLUOLYHUV
ZHUH H[FLVHG *)3 WXPRUV ZHUH YLVXDOL]HG DQG TXDQWLILHG XVLQJ DQ HSLIOXRUHVFHQFH GLVVHFWLRQ
VFRSH 1LNRQ 7LVVXHV ZHUH IL[HG LQ SDUDIRUPDOGHK\GH DQG VWDLQHG ZLWK KHPDWR[\OLQ DQG
HRVLQIROORZLQJSDUDIILQHPEHGGLQJ
,PPXQRKLVWRFKHPLFDODQG,PPXQRF\WRFKHPLFDO$QDO\VLV
)ROORZLQJ QHFURSV\ WXPRUV DQG OLYHUV ZHUH IL[HG RYHUQLJKW LQ SDUDIRUPDOGHK\GH DW ž&
7LVVXHV XVHG IRU SDUDIILQ HPEHGGLQJ ZHUH WUDQVIHUUHG WR FDVVHWWHV DQG SODFHG LQ HWKDQRO
7KRVHXVHGIRUIUR]HQVHFWLRQVZHUHSODFHGLQVXFURVHRYHUQLJKWDWž&7LVVXHVZHUHWKHQ
WUDQVIHUUHG WR 2&7 FRPSRXQG 7LVVXH7HN IRU KRXUV DW URRP WHPSHUDWXUH IROORZHG E\
IUHH]LQJ LQ LVRSHQWDQH 6LJPD SODFHG LQ D OLTXLG QLWURJHQ EDWK &RVWDLQLQJ RI JDOHFWLQ DQG
)ZDVSHUIRUPHGRQIUR]HQVHFWLRQVXVLQJDQWLERG\FORQHV0%LR/HJHQGDQG&,$
%LR/HJHQGUHVSHFWLYHO\1XFOHLZHUHVWDLQHGZLWK+RHFKVW,QYLWURJHQ)RUVWXGLHVRIOLYHUV
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
SULRU WR WKH GHWHFWLRQ RI RYHUW PHWDVWDVHV H[FLVHG WLVVXHV ZHUH H[DPLQHG JURVVO\ IRU WKH
DSSHDUDQFHRIQRGXOHVDQGPLFURVFRSLFDOO\IROORZLQJVWDLQLQJIRUWKHSUHVHQFHRIWXPRUV
)RU IOXRUHVFHQW OHFWLQ DQDO\VLV RI 7$QWLJHQ SUHVHQWDWLRQ FHOOV ZHUH VWDLQHG ZLWK 31$
$OH[D)OXRU LQYLWURJHQ $OO SDUDIILQ VHFWLRQV ZHUH GHYHORSHG XVLQJ '$% DQG
FRXQWHUVWDLQHGZLWKKHPDWR[\OLQ&DUER)UHH%ORFNLQJ6ROXWLRQ9HFWRU/DERUDWRULHVZDVXVHG
IRU DOO VWDLQLQJ LQYROYLQJ WKH XVH RI SHDQXW DJJOXWLQLQ (QGRJHQRXV SHUR[LGDVH DFWLYLW\ ZDV
EORFNHG XVLQJ WKH 'XDO (QGRJHQRXV (Q]\PH %ORFN 'DNR 31$ VWDLQLQJ RI KXPDQ WLVVXH
PLFURDUUD\V ZDV SHUIRUPHG ZLWK 31$+53 6LJPD —JP/ IROORZLQJ DQWLJHQ UHWULHYDO LQ
FLWUDWHEXIIHU6WDLQLQJRIWKH+XPDQ/XQJ&DQFHUDQG1RUPDO7LVVXH0LFURDUUD\86%LRPD[
,QFZDVVFRUHGIRUWKHSUHVHQFHRIVXUIDFH7$QWLJHQVWDLQLQJLQDEOLQGHGIDVKLRQ
$OO LPDJHV ZHUH DFTXLUHG XVLQJ DQ LQYHUWHG 1LNRQ 7L( HSLIOXRUHVFHQFH PLFURVFRSH XVLQJ
(OHPHQWV VRIWZDUH 1LNRQ 3VHXGRFRORULQJ ZDV SHUIRUPHG XVLQJ (OHPHQWV RU ,PDJH- 1,+
4XDQWLILFDWLRQRIKLVWRORJ\ZDVSHUIRUPHGXVLQJ(OHPHQWV
:HVWHUQDQG31$/HFWLQ%ORW$QDO\VLV
&HOO O\VDWHV ZHUH KDUYHVWHG LQ 5,3$ EXIIHU 6LJPD FRQWDLQLQJ &RPSOHWH 3URWHDVH ,QKLELWRU
5RFKHDQG3KRVSKR6WRS5RFKHRQLFHDQGUXQRQ6'63$*(JHOV39')PHPEUDQHVZHUH
VWDLQHG IRU JDOHFWLQ $EFDP DE RU %LR/HJHQG 0 JDOHFWLQ
$EFDP DE DQG ĮWXEXOLQ &HOO 6LJQDOLQJ /HFWLQ EORWWLQJ IRU
JO\FDQV ZDV SHUIRUPHG XVLQJ 31$%LRWLQ 6LJPD IROORZHG E\ GHWHFWLRQ XVLQJ WKH $%& (OLWH
.LW 9HFWRU /DEV 3. RU 31$3HUR[LGDVH FRQMXJDWHV 6LJPD 3RQFHDX 6 VROXWLRQ
7KHUPRZDVXVHGIRUWRWDOSURWHLQGHWHFWLRQIROORZLQJWUDQVIHUWR39')PHPEUDQHV
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
)RU OLYHU KRPRJHQDWH ZHVWHUQ EORWV PLFH EHDULQJ 0 IODQN WXPRUV RU QR WXPRUV ZHUH
SHUIXVHG E\ LQWUDFDUGLDF LQMHFWLRQ RI P/ RI SKRVSKDWH EXIIHUHG VDOLQH /LYHUV ZHUH WKHQ
H[FLVHGDQGPJSRUWLRQVZHUHDGGHGWRJHQWOH0$&60WXEHV0LOWHQ\L%LRWHFKLQP/RI
5,3$ 6LJPD ZLWK &RPSOHWH 3URWHDVH ,QKLELWRUV 5RFKH 7LVVXH ZDV GLVVRFLDWHG XVLQJ WKH
JHQWOH0$&62FWR'LVVRFLDWRU0LOWHQ\L%LRWHFKDQGUXQRQSRO\DFU\ODPLGHJHOVDVGHVFULEHG
DERYH
*DOHFWLQ(/,6$
3HULSKHUDOEORRGZDVKDUYHVWHGIURPPLFHZLWKRXWWXPRUVRUEHDULQJFRQWUDODWHUDOIODQNWXPRUV
RIERWK7DQG7FHOOOLQHVE\FDUGLDFSXQFWXUHDVGHVFULEHGDERYH%ORRGZDVFROOHFWHG
LQ VHUXP FROOHFWLRQ WXEHV &DSLMHFW DQG DOORZHG WR FORW IRU PLQXWHV SULRU WR FHQWULIXJDWLRQ
DQG UHPRYDO RI VHUXP 7KH JDOHFWLQ (/,6$ NLW 5' 6\VWHPV ZDV XVHG DFFRUGLQJ WR WKH
PDQXIDFWXUHU¶VLQVWUXFWLRQV
&HOO6XUIDFH3URWHLQ,VRODWLRQ
&HOO VXUIDFH SURWHLQV ZHUH LVRODWHG SULRU WR :HVWHUQ EORW DQDO\VLV XVLQJ 7KHUPR 6FLHQWLILF NLW
IROORZLQJ WKH PDQXIDFWXUHU¶V LQVWUXFWLRQV %ULHIO\ FHOOV DUH ZDVKHG IROORZHG E\
ELRWLQ\ODWLRQRIWKHVXUIDFHSURWHLQVE\D6XOIR1+6%LRWLQWKURXJKFRQMXJDWLRQWRIUHHDPLQHV
7KHUHDFWLRQLVTXHQFKHGDQGFHOOVDUHKDUYHVWHGDQGO\VHG/\VDWHLVZDVKHGWKURXJKDFROXPQ
FRQWDLQLQJDQHXWUDYLGLQUHVLQWRFDSWXUHWKHELRWLQ\ODWHGVXUIDFHSURWHLQV)LQDOO\WKHGLVXOILGH
OLQNDJHLVUHGXFHGWKURXJKLQFXEDWLRQZLWKP0'77DQGWKHSURWHLQVDUHFDSWXUHG
)ORZ&\WRPHWULF$QDO\VLV
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
3HULSKHUDOEORRGZDVGUDZQE\FDUGLDFSXQFWXUHDQGWUDQVIHUUHGWR.('7$PJ3OXV%ORRG
&ROOHFWLRQ7XEHV%'9DFXWDLQHU%'%LRVFLHQFHV5HGEORRGFHOOVZHUHO\VHGXVLQJ5%&O\VLV
EXIIHU H%LRVFLHQFHV &HOOV ZHUH IL[HG LQ SDUDIRUPDOGHK\GH DQG ZDVKHG SULRU WR VWDLQLQJ
&HOOV ZHUH EORFNHG XVLQJ DQWLPRXVH &'&' DQWLERG\ FORQH * %' %LRVFLHQFHV
$QWLERGLHV DJDLQVW PRXVH DQWLJHQV ZHUH &'E 0 %' %LRVFLHQFHV &' $)6
H%LRVFLHQFHV 0DF 0 %LR/HJHQG /\& +. %LR/HJHQG DQG *U 5%&
%LR/HJHQG)ORZF\WRPHWU\ZDVSHUIRUPHGRQD/65)RUWHVVD%'%LRVFLHQFHVDQGDQDO\]HG
XVLQJ)ORZMR7UHH6WDU
0DJQHWLF VHSDUDWLRQ RI &'E FHOOV ZDV SHUIRUPHG XVLQJ &'E 0$&6 PLFUREHDGV
0LOWHQ\L )ROORZLQJ FROOHFWLRQ RI SHULSKHUDO EORRG E\ FDUGLDF SXQFWXUH DQG O\VLV RI
HU\WKURF\WHV FHOOV ZHUH LQFXEDWHG ZLWK WKH 0$&6 EHDGV IROORZLQJ WKH PDQXIDFWXUHU¶V
LQVWUXFWLRQV&'EFHOOVZHUHLVRODWHGXVLQJ0$&606&ROOHFWLRQ&ROXPQV0LOWHQ\L
3HDQXW DJJOXWLQLQ VWDLQLQJ ZDV SHUIRUPHG E\ LQFXEDWLQJ î FHOOV LQ 3%6 ZLWK —/ 31$
$OH[D)OXRU LQYLWURJHQ RQ LFH IRU PLQXWHV )OXRUHVFHQW JDOHFWLQ ZDV SURGXFHG E\
UHDFWLQJUHFRPELQDQWPXULQHJDOHFWLQ5'6\VWHPVZLWK'\/LJKW1+6(VWHU7KHUPR
3LHUFH &RQMXJDWHG SURWHLQ ZDV SXULILHG E\ )3/& DQG FRQFHQWUDWHG WR PJP/ XVLQJ 0:&2$PLFRQFHQWULIXJDOILOWHUXQLWV0LOOLSRUH&HOOVZHUHVWDLQHGE\LQFXEDWLQJîFHOOV
LQ3%6ZLWK—/RIWKHIOXRUHVFHQWJDOHFWLQRQLFHIRUPLQXWHV
)RUJDOHFWLQELQGLQJLQKLELWLRQH[SHULPHQWVRQWXPRUFHOOVFHOOVZHUHLQFXEDWHGZLWKJDOHFWLQ
DVGHVFULEHGDERYHLQWKHSUHVHQFHRIP0N$FHW\O'ODFWRVDPLQH&DUERV\QWKRUVXFURVH
6LJPD )RU FRPSHWLWLRQ RI JDOHFWLQ SUHVHQWDWLRQ RQ OHXNRF\WHV SHULSKHUDO EORRG ZDV
KDUYHVWHGIURPPLFHEHDULQJ0IODQNWXPRUVDVGHVFULEHGDERYH&HOOVZHUHLQFXEDWHGZLWK
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
P0ȕODFWRVH6LJPDRUVXFURVH6LJPDLQ)%6IRUPLQXWHVRQLFH&HOOVZHUHWKHQ
IL[HGVWDLQHGDQGDQDO\]HGE\IORZF\WRPHWU\
&RQGLWLRQHG0HGLD([SHULPHQWV
7ZHQW\ILYH PLOOLOLWHUV RI FRQGLWLRQHG PHGLXP ZDV KDUYHVWHG IURP RQH 7 IODVN RI 0V
IROORZLQJWKUHHGD\VRIFXOWXUH7KLVPHGLXPRUIUHVKFRQWUROPHGLXPZLWKVHUXPZDVILOWHUHG
WKURXJK—PILOWHUVDQGFRQFHQWUDWHGIROGXVLQJ0:&2FHQWULIXJDOILOWHUV$PLFRQ
—/ RI PHGLXP ZDV LQMHFWHG LQWR WKH ODWHUDO WDLO YHLQ RI PLFH 7ZR KRXUV DIWHU LQMHFWLRQV
SHULSKHUDOEORRGZDVKDUYHVWHGIURPWKHPLFHE\FDUGLDFSXQFWXUHDVGHVFULEHGDERYHIROORZHG
E\ IL[DWLRQ VWDLQLQJ DQG IORZ F\WRPHWULF DQDO\VLV )RU H[SHULPHQWV LQYROYLQJ UHFRPELQDQW
JDOHFWLQPXULQHUHFRPELQDQWJDOHFWLQ5'6\VWHPVZDVVXSSOHPHQWHGWRWKHPHGLXPDW
—J SHU LQMHFWLRQ )RU ,/ H[SHULPHQWV PXULQH UHFRPELQDQW ,/ 5' 6\VWHPV ZDV
VXSSOHPHQWHGWRWKHPHGLXPDWQJSHULQMHFWLRQ
/XPLQH[&\WRNLQH3URILOLQJ
&\WRNLQH SURILOHV RI X/ FHOO FXOWXUH VXSHUQDWDQW IURP FRQWURO DQG FRQGLWLRQHG PHGLD ZHUH
DVVHVVHG XVLQJ D (0' 0LOOLSRUH 0LOOLSOH[ FXVWRP PDJQHWLF EHDG SDQHO XVLQJ PDQXIDFWXUHU¶V
SURWRFRO ,/ ,)1J ,/ 71)D ,/D ,/ ,/ ,/ /XPLQH[ DVVD\V ZHUH DQDO\]HG
XVLQJ%LRSOH[ZLWK%LRSOH[0DQDJHU
51$,VRODWLRQDQG([SUHVVLRQ3URILOLQJ
0XULQH JHQH H[SUHVVLRQ PLFURDUUD\ DQDO\VLV ZDV GHVFULEHG SUHYLRXVO\ DQG LV DYDLODEOH
IURP 1&%, XQGHU DFFHVVLRQ QXPEHU *6( DQG *6( +XPDQ 16&/& H[SUHVVLRQ
PLFURDUUD\GDWDZDVDFFHVVHGIURP1&%,DFFHVVLRQQXPEHU*6(51$ZDVLVRODWHGXVLQJ
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
51HDV\ PLQL NLWV 4LDJHQ DFFRUGLQJ WR WKH PDQXIDFWXUHU¶V LQVWUXFWLRQV F'1$ UHDFWLRQV ZHUH
SHUIRUPHG XVLQJ L6FULSW F'1$ V\QWKHVLV NLW %LR5DG T3&5 UHDFWLRQV ZHUH SHUIRUPHG XVLQJ
—/RIF'1$—/RI,46<%5*UHHQ6XSHUPL[%LR5DGDQG—PRORIHDFKSULPHU)ROG
FKDQJH LV UHSRUWHG IROORZLQJ DQDO\VLV XVLQJ WKHǻǻ&W PHWKRG ZKHUH JHQHV ZHUH QRUPDOL]HG WR
Hprt
KRXVHNHHSLQJ
FRQWURO
3ULPHUV
ZHUH
St6galnac4
IRUZDUG
¶
**77**77&$&&$7*$77&7*¶ St6galnac4 UHYHUVH ¶**$*&****$&7&77&7&¶
Gcnt3
IRUZDUG
¶*&$*&&$$*$$**7$&&$$$¶
Gcnt3
UHYHUVH
¶
$&$**&*$**$&&$7&$$¶ RU IRUZDUG ¶$7*$*$*&&$77*&*$*$&7¶ UHYHUVH
¶&77*77**&&$$*$**7*$7¶ Hprt IRUZDUG ¶*7&$$&*****$&$7$$$$*¶
Hprt UHYHUVH ¶&$$&$$7&$$*$&$77&777$&$¶ *HQH H[SUHVVLRQ DQDO\VLV DQG
YLVXDOL]DWLRQVZHUHSHUIRUPHGXVLQJ6SRWILUH7LEFRDQG0$7/$%0DWKZRUNV
+XPDQ&RS\1XPEHU$QDO\VLV
&RS\QXPEHUGHWHUPLQDWLRQVRIGCNT3DQGIL6ZHUHSHUIRUPHGXVLQJ2QFRPLQHEDVHGRQ
SXEOLFO\DYDLODEOHGDWDIURPWKH&DQFHU*HQRPH$WODV
+DLUSLQVDQG3ODVPLGV
.QRFNGRZQ
RI
6WJDOQDF
ZDV
SHUIRUPHG
XVLQJ
VKRUWKDLUSLQ
51$
¶
77&7*&7&&7&$&$&7*7*&$7&77*$&$¶ RU D FRQWURO KDLUSLQ WDUJHWLQJ ILUHIO\
OXFLIHUDVH LQ WKH S*)3956 YHFWRU 2ULJHQH 7* DQG 75 3ODVPLGV ZHUH
SDFNDJHG LQWR UHWURYLUXVHV XVLQJ WKH 3KRHQL[(FR V\VWHP 9LUDO VXSHUQDWDQW ZDV ILOWHUHG XVLQJ
—P V\ULQJH ILOWHUV DQG FXOWXUHG ZLWK 0 FHOOV DW QR GLOXWLRQ 7UDQVGXFHG FHOOV ZHUH
VHOHFWHGZLWKSXURP\FLQ,QYLWURJHQDQGNQRFNGRZQHIILFLHQF\ZDVGHWHUPLQHGE\T573&5
IROORZLQJ IRXU ZHHNV RI FXOWXUH 7DQVIHFWLRQ RI Gcnt3 ZDV SHUIRUPHG XVLQJ /LSRIHFWDPLQH
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
,QYLWURJHQDFFRUGLQJWRWKHPDQXIDFWXUHU¶VLQVWUXFWLRQVZLWKSODVPLGVSXUFKDVHGIURP2ULJHQH
0& .QRFNGRZQ RI /JDOV ZDV SHUIRUPHG XVLQJ WZR VKRUWKDLUSLQ 51$V 6LJPD
VK*DO
¶
&&***&$*7$&$$&&$7&**$7*$$&7&*$*77&$7&&*$7**77*7$&7*&77777
*¶
VK*DO
¶
&&**&&*&$7*&7*$7&$&$$7&$7&7&*$*$7*$77*7*$7&$*&$7*&**77777
*¶ RU D FRQWURO KDLUSLQ WDUJHWLQJ ILUHIO\ OXFLIHUDVH LQ 6LJPD 6+&9 $GGLWLRQDOO\ D
VHFRQG KDLUSLQ V\VWHP ZDV XVHG WR NQRFNGRZQ St6galnac4 IURP 6LJPD VK6WJDOQDF
&&***7$&$&&77&$&7*$$&*&$7&7&*$*$7*&*77&$*7*$$**7*7$&77777
*
In vitro 3UROLIHUDWLRQ$VVD\V
&HOOVH[SUHVVLQJKDLUSLQVDJDLQVWSt6galnac4RUFRQWUROKDLUSLQVZHUHVHHGHGLQWRZHOOSODWHV
1HZZHOOVZHUHWU\SVLQL]HGDQGFRXQWHGXVLQJDKHPRF\WRPHWHUZLWK7U\SDQ%OXHH[FOXVLRQDW
KRXULQWHUYDOV
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
$XWKRUV¶&RQWULEXWLRQV
6 %KDWLD DQG 1 5HWLFNHU)O\QQ GHVLJQHG WKH VWXGLHV DQG ZURWH WKH PDQXVFULSW 1 5HWLFNHU
)O\QQSHUIRUPHGDOOH[SHULPHQWVDQGGDWDDQDO\VLV
*UDQW6XSSRUW
7KLVZRUNZDVVXSSRUWHGLQSDUWE\WKH.RFK,QVWLWXWH6XSSRUWFRUH*UDQW3&$IURP
WKH1DWLRQDO&DQFHU,QVWLWXWH15HWLFNHU)O\QQLVVXSSRUWHGE\D/XGZLJ&HQWHUIRU0ROHFXODU
2QFRORJ\*UDGXDWH)HOORZVKLS6%KDWLDLVDQ++0,LQYHVWLJDWRU7KLVZRUNZDVVXSSRUWHGE\
D6WDQG8S7R&DQFHU'UHDP7HDP7UDQVODWLRQDO5HVHDUFK*UDQW*UDQW1XPEHU68&$$&5
'7 6WDQG 8S 7R &DQFHU LV D SURJUDP RI WKH (QWHUWDLQPHQW ,QGXVWU\ )RXQGDWLRQ
DGPLQLVWHUHG E\ WKH $PHULFDQ $VVRFLDWLRQ IRU &DQFHU 5HVHDUFK +RZDUG +XJKHV 0HGLFDO
,QVWLWXWHDQGWKH.RFK&7&3URMHFW
$FNQRZOHGJHPHQWV
7KH DXWKRUV WKDQN 5 :HLQEHUJ DQG + )OHPLQJ IRU FULWLFDO UHDGLQJ RI WKH PDQXVFULSW 7KH\
WKDQN5+\QHV-/RYH'+DEHU16WHEELQV&6XOOLYDQDQGDOOPHPEHUVRIWKH6%KDWLD
ODEIRUWKHLULQVLJKWIXOGLVFXVVLRQV7KH\WKDQN0:LQVORZDQG7-DFNVIRUSURYLGLQJWKHFHOO
OLQHV DQG JHQHUDO LQVLJKW 7KH\ WKDQN WKH .RFK ,QVWLWXWH 6ZDQVRQ %LRWHFKQRORJ\ &HQWHU &RUH
)DFLOLWLHVIRUWHFKQLFDOVXSSRUWVSHFLILFDOO\ZLWKWLVVXHVHFWLRQLQJDQGXVDJHRIIORZF\WRPHWHUV
7KH\ WKDQN WKH 7&*$ DQG DFNQRZOHGJH XVH RI WKH 7&*$ 'DWD 3RUWDO 7KH DXWKRUV ZLVK WR
GHGLFDWHWKLVPDQXVFULSWWRWKHPHPRU\RI2IILFHU6HDQ&ROOLHUIRUKLVFDULQJVHUYLFHWRWKH0,7
FRPPXQLW\DQGIRUKLVVDFULILFH
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
5HIHUHQFHV
/LX )7 5DELQRYLFK *$ *DOHFWLQV DV PRGXODWRUV RI WXPRXU SURJUHVVLRQ 1DW 5HY &DQFHU
$OPNYLVW-.DUOVVRQ$*DOHFWLQVDVLQIODPPDWRU\PHGLDWRUV*O\FRFRQM-
5DELQRYLFK *$ 7RVFDQR 0$ 7XUQLQJ VZHHW
RQ LPPXQLW\ JDOHFWLQJO\FDQ LQWHUDFWLRQV LQ
LPPXQHWROHUDQFHDQGLQIODPPDWLRQ1DW5HY,PPXQRO
,XULVFL,7LQDUL11DWROL&$QJHOXFFL'&LDQFKHWWL(,DFREHOOL6&RQFHQWUDWLRQVRI*DOHFWLQ
LQWKH6HUDRI1RUPDO&RQWUROVDQG&DQFHU3DWLHQWV&OLQLFDO&DQFHU5HVHDUFK
%DODQ91DQJLD0DNNHU35D]$*DOHFWLQVDV&DQFHU%LRPDUNHUV&DQFHUV
-RKQ &0 /HIIOHU + .DKO.QXWVVRQ % 6YHQVVRQ , -DUYLV *$ 7UXQFDWHG *DOHFWLQ ,QKLELWV
7XPRU *URZWK DQG 0HWDVWDVLV LQ 2UWKRWRSLF 1XGH 0RXVH 0RGHO RI +XPDQ %UHDVW &DQFHU &OLQLFDO
&DQFHU5HVHDUFK
*OLQVN\99*OLQVN\*95LWWHQKRXVH2OVRQ.+XIOHMW0(*OLQVNLL29'HXWVFKHU6/HWDO
7KH5ROHRI7KRPVHQ)ULHGHQUHLFK$QWLJHQLQ$GKHVLRQRI+XPDQ%UHDVWDQG3URVWDWH&DQFHU&HOOVWR
WKH(QGRWKHOLXP&DQFHU5HVHDUFK
%LDQ&)=KDQJ<6XQ+/L'):DQJ'&6WUXFWXUDOEDVLVIRUGLVWLQFWELQGLQJSURSHUWLHVRI
WKHKXPDQJDOHFWLQVWR7KRPVHQ)ULHGHQUHLFKDQWLJHQ3/R621(H
<X /* 7KH RQFRIHWDO 7KRPVHQ±)ULHGHQUHLFK FDUERK\GUDWH DQWLJHQ LQ FDQFHU SURJUHVVLRQ
*O\FRFRQMXJDWH-RXUQDO
6SULQJHU *) ,PPXQRUHDFWLYH 7 DQG 7Q HSLWRSHV LQ FDQFHU GLDJQRVLV SURJQRVLV DQG
LPPXQRWKHUDS\-0RO0HG%HUO
-R\FH -$ 3ROODUG -: 0LFURHQYLURQPHQWDO UHJXODWLRQ RI PHWDVWDVLV 1DW 5HY &DQFHU
3VDLOD%/\GHQ'7KHPHWDVWDWLFQLFKHDGDSWLQJWKHIRUHLJQVRLO1DW5HY&DQFHU
3ROODUG-:7XPRXUHGXFDWHGPDFURSKDJHVSURPRWHWXPRXUSURJUHVVLRQDQGPHWDVWDVLV1DW5HY
&DQFHU
.DSODQ515LED5'=DFKDURXOLV6%UDPOH\$+9LQFHQW/&RVWD&HWDO9(*)5SRVLWLYH
KDHPDWRSRLHWLFERQHPDUURZSURJHQLWRUVLQLWLDWHWKHSUHPHWDVWDWLFQLFKH1DWXUH
(UOHU -7 %HQQHZLWK ./ &R[ 75 /DQJ * %LUG ' .RRQJ $ HW DO +\SR[LD,QGXFHG /\V\O
2[LGDVH ,V D &ULWLFDO 0HGLDWRU RI %RQH 0DUURZ &HOO 5HFUXLWPHQW WR )RUP WKH 3UHPHWDVWDWLF 1LFKH
&DQFHU&HOO
2VNDUVVRQ 7 $FKDU\\D 6 =KDQJ ;+) 9DQKDUDQWD 6 7DYD]RLH 6) 0RUULV 3* HW DO %UHDVW
FDQFHU FHOOV SURGXFH WHQDVFLQ & DV D PHWDVWDWLF QLFKH FRPSRQHQW WR FRORQL]H WKH OXQJV 1DW 0HG
0F$OOLVWHU 66 *LIIRUG $0 *UHLQHU $/ .HOOHKHU 63 6DHO]OHU 03 ,QFH 7$ HW DO 6\VWHPLF
(QGRFULQH,QVWLJDWLRQRI,QGROHQW7XPRU*URZWK5HTXLUHV2VWHRSRQWLQ&HOO
0DODQFKL , 6DQWDPDULD0DUWLQH] $ 6XVDQWR ( 3HQJ + /HKU +$ 'HODOR\H -) HW DO
,QWHUDFWLRQV EHWZHHQ FDQFHU VWHP FHOOV DQG WKHLU QLFKH JRYHUQ PHWDVWDWLF FRORQL]DWLRQ 1DWXUH
.LP67DNDKDVKL+/LQ::'HVFDUJXHV3*ULYHQQLNRY6.LP<HWDO&DUFLQRPDSURGXFHG
IDFWRUVDFWLYDWHP\HORLGFHOOVWKURXJK7/5WRVWLPXODWHPHWDVWDVLV1DWXUH
'X5/X.93HWULWVFK&/LX3*DQVV53DVVHJXp(HWDO+,)Į,QGXFHVWKH5HFUXLWPHQWRI
%RQH0DUURZ'HULYHG9DVFXODU0RGXODWRU\&HOOVWR5HJXODWH7XPRU$QJLRJHQHVLVDQG,QYDVLRQ&DQFHU
&HOO
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
.LP 0< 2VNDUVVRQ 7 $FKDU\\D 6 1JX\HQ '; =KDQJ ;+) 1RUWRQ / HW DO 7XPRU 6HOI
6HHGLQJE\&LUFXODWLQJ&DQFHU&HOOV&HOO
.DQJ < 6LHJHO 30 6KX : 'UREQMDN 0 .DNRQHQ 60 &RUGyQ&DUGR & HW DO $ PXOWLJHQLF
SURJUDPPHGLDWLQJEUHDVWFDQFHUPHWDVWDVLVWRERQH&DQFHU&HOO
0LQQ$-*XSWD*36LHJHO30%RV3'6KX:*LUL''HWDO*HQHVWKDWPHGLDWHEUHDVWFDQFHU
PHWDVWDVLVWROXQJ1DWXUH
%RV3'=KDQJ;+)1DGDO&6KX:*RPLV551JX\HQ';HWDO*HQHVWKDWPHGLDWHEUHDVW
FDQFHUPHWDVWDVLVWRWKHEUDLQ1DWXUH
4LDQ%=/L-=KDQJ+.LWDPXUD7=KDQJ-&DPSLRQ/5HWDO&&/UHFUXLWVLQIODPPDWRU\
PRQRF\WHVWRIDFLOLWDWHEUHDVWWXPRXUPHWDVWDVLV1DWXUH
<DQJ/+XDQJ-5HQ;*RUVND$(&K\WLO$$DNUH0HWDO$EURJDWLRQRI7*)EHWDVLJQDOLQJ
LQ PDPPDU\ FDUFLQRPDV UHFUXLWV *U&'E P\HORLG FHOOV WKDW SURPRWH PHWDVWDVLV &DQFHU &HOO
-DFNVRQ (/ :LOOLV 1 0HUFHU . %URQVRQ 57 &URZOH\ ' 0RQWR\D 5 HW DO $QDO\VLV RI OXQJ
WXPRULQLWLDWLRQDQGSURJUHVVLRQXVLQJFRQGLWLRQDOH[SUHVVLRQRIRQFRJHQLF.UDV*HQHV'HYHORSPHQW
:LQVORZ 00 'D\WRQ 7/ 9HUKDDN 5*: .LP.LVHODN & 6Q\GHU (/ )HOGVHU '0 HW DO
6XSSUHVVLRQRIOXQJDGHQRFDUFLQRPDSURJUHVVLRQE\1N[1DWXUH
'X3DJH 0 &KHXQJ $) 0D]XPGDU & :LQVORZ 00 %URQVRQ 5 6FKPLGW /0 HW DO
(QGRJHQRXV7&HOO5HVSRQVHVWR$QWLJHQV([SUHVVHGLQ/XQJ$GHQRFDUFLQRPDV'HOD\0DOLJQDQW7XPRU
3URJUHVVLRQ&DQFHU&HOO
5HWLFNHU)O\QQ 1( 0DOWD ')% :LQVORZ 00 /DPDU -0 ;X 0- 8QGHUKLOO *+ HW DO $
FRPELQDWRULDO H[WUDFHOOXODU PDWUL[ SODWIRUP LGHQWLILHV FHOOH[WUDFHOOXODU PDWUL[ LQWHUDFWLRQV WKDW FRUUHODWH
ZLWKPHWDVWDVLV1DW&RPPXQ
'RQJ 6 +XJKHV 5& 0DFURSKDJH VXUIDFH JO\FRSURWHLQV ELQGLQJ WR JDOHFWLQ 0DFDQWLJHQ
*O\FRFRQM-
+LUDWVXND 6 :DWDQDEH $ $EXUDWDQL + 0DUX < 7XPRXUPHGLDWHG XSUHJXODWLRQ RI
FKHPRDWWUDFWDQWV DQG UHFUXLWPHQW RI P\HORLG FHOOV SUHGHWHUPLQHV OXQJ PHWDVWDVLV 1DW &HOO %LRO
6DQR + +XPDQ JDOHFWLQ LV D QRYHO FKHPRDWWUDFWDQW IRU PRQRF\WHV DQG PDFURSKDJHV -
,PPXQRO
6DWR 6 2XHOOHW 1 3HOOHWLHU , 6LPDUG 0 5DQFRXUW $ %HUJHURQ 0* 5ROH RI JDOHFWLQ DV DQ
DGKHVLRQ PROHFXOH IRU QHXWURSKLO H[WUDYDVDWLRQ GXULQJ VWUHSWRFRFFDO SQHXPRQLD - ,PPXQRO
)XQFWLRQDO *O\FRPLFV *DWHZD\ &RQVRUWLXP IRU )XQFWLRQDO *O\FRPLFV
KWWSZZZIXQFWLRQDOJO\FRPLFVRUJ
6SULQJHU*)7DQG7QJHQHUDOFDUFLQRPDDXWRDQWLJHQV6FLHQFH
+VX<&<XDQ6&KHQ+<<X 6//LX&++VX3<HWDO$)RXU*HQH6LJQDWXUHIURP
1&, &HOO /LQH IRU 6XUYLYDO 3UHGLFWLRQ LQ 1RQ±6PDOO &HOO /XQJ &DQFHU &OLQLFDO &DQFHU 5HVHDUFK
,QND%3DWKZD\VRI2JO\FDQELRV\QWKHVLVLQFDQFHUFHOOV%LRFKLPLFDHW%LRSK\VLFD$FWD%%$
*HQHUDO6XEMHFWV
+VHPDQQ<*HLJO-%6FKXEHUW)0XVLDQL30H\HU0%XUJKDUW(HWDO6\VWHPLF6SUHDG,V
DQ(DUO\6WHSLQ%UHDVW&DQFHU&DQFHU&HOO
&RXVVHQV/0:HUE=,QIODPPDWLRQDQGFDQFHU1DWXUH
1DQJLD0DNNHU 3 *DOHFWLQ LQGXFHV HQGRWKHWLDO FHOO PRUSKRJHQHVLV DQG DQJLRJHQHVLV $P -
3DWKRO
8HGD 7 6KLPDGD ( 8UDNDZD 7 6HUXP OHYHOV RI F\WRNLQHV LQ SDWLHQWV ZLWK FRORUHFWDO FDQFHU
3RVVLEOH LQYROYHPHQW RI LQWHUOHXNLQ DQG LQWHUOHXNLQ LQ KHPDWRJHQRXV PHWDVWDVLV - *DVWURHQWHURO
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
%OD\ -< 1HJULHU 6 &RPEDUHW 9 $WWDOL 6 *RLOORW ( 0HUURXFKH < HW DO 6HUXP /HYHO RI
,QWHUOHXNLQDVD3URJQRVLV)DFWRULQ0HWDVWDWLF5HQDO&HOO&DUFLQRPD&DQFHU5HVHDUFK
1DNDVKLPD - 7DFKLEDQD 0 +RULJXFKL < 2\D 0 2KLJDVKL 7 $VDNXUD + HW DO 6HUXP
,QWHUOHXNLQ DV D 3URJQRVWLF )DFWRU LQ 3DWLHQWV ZLWK 3URVWDWH &DQFHU &OLQLFDO &DQFHU 5HVHDUFK
%XUJHU006XUIDFHFKDQJHVLQWUDQVIRUPHGFHOOVGHWHFWHGE\OHFWLQV)HG3URF
.LP < 9DUNL $ 3HUVSHFWLYHV RQ WKH VLJQLILFDQFH RI DOWHUHG JO\FRV\ODWLRQ RI JO\FRSURWHLQV LQ
FDQFHU*O\FRFRQMXJDWH-RXUQDO
+DNRPRUL6*O\FRV\ODWLRQGHILQLQJFDQFHUPDOLJQDQF\1HZZLQHLQDQROGERWWOH3URFHHGLQJV
RIWKH1DWLRQDO$FDGHP\RI6FLHQFHV
8HGD.*O\FRSURWHRPLFVWUDWHJLHV)URPGLVFRYHU\WRFOLQLFDODSSOLFDWLRQRIFDQFHUFDUERK\GUDWH
ELRPDUNHUV3527(20,&6±&OLQLFDO$SSOLFDWLRQV
.XIH':0XFLQVLQFDQFHUIXQFWLRQSURJQRVLVDQGWKHUDS\1DW5HY&DQFHU
/DNVKPLQDUD\DQDQ 9 7KRPSVRQ 3 :ROIHUW 0$ %XVNDV 7 %UDGOH\ -0 3DWKDQJH\ /% HW DO
,PPXQH UHFRJQLWLRQ RI WXPRUDVVRFLDWHG PXFLQ 08& LV DFKLHYHG E\ D IXOO\ V\QWKHWLF DEHUUDQWO\
JO\FRV\ODWHG08&WULSDUWLWHYDFFLQH3URF1DWO$FDG6FL86$
<RJHHVZDUDQ*6DON3/0HWDVWDWLFSRWHQWLDOLVSRVLWLYHO\FRUUHODWHGZLWKFHOOVXUIDFHVLDO\ODWLRQ
RIFXOWXUHGPXULQHWXPRUFHOOOLQHV6FLHQFH
0XUXJDHVX1,UDYDQL0YDQ:HYHUZLMN$,YHWLF$-RKQVRQ'$$QWRQRSRXORV$HWDO$Q,Q
9LYR )XQFWLRQDO 6FUHHQ ,GHQWLILHV 67*DO1$F 6LDO\OWUDQVIHUDVH DV D %UHDVW &DQFHU 0HWDVWDVLV
6XSSUHVVRU&DQFHU'LVFRYHU\
5KRGHV '5 <X - 6KDQNHU . 'HVKSDQGH 1 9DUDPEDOO\ 5 *KRVK ' HW DO 21&20,1( D
FDQFHUPLFURDUUD\GDWDEDVHDQGLQWHJUDWHGGDWDPLQLQJSODWIRUP1HRSODVLD
7KH&DQFHU*HQRPH$WODVKWWSVWFJDGDWDQFLQLKJRYWFJDWFJD+RPHMVS
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
)LJXUH/HJHQGV
)LJXUHMice bearing tumors have accumulation of the adhesion ligand, galectin-3, in the early
metastatic niche $ &HOO OLQHV DUH JHQHUDWHG IURP DXWRFKWKRQRXV WXPRUV LQ KrasLSLG12D/+
;p53flox/floxPLFHIROORZLQJWXPRUGHYHORSPHQW7KHOLQHVZKLFKUHSUHVHQWGLVFUHWHVWDJHVRI
PHWDVWDVLV DUH GHULYHG IURP SULPDU\ WXPRUV WKDW GLG QRW PHWDVWDVL]H 7QRQ0HW SULPDU\ WXPRUV
WKDWJDYHULVHWRPHWDVWDVHV70HWO\PSKQRGHPHWDVWDVHV1QRWVKRZQDQGGLVWDQWPHWDVWDVHV
0/LQHVGHULYHGIURPLQFUHDVLQJO\PHWDVWDWLFWXPRUVH[KLELWLQFUHDVHGDGKHVLRQWRJDOHFWLQ
DQG JDOHFWLQ % 0LFH EHDULQJ WKH QR WXPRUV 7QRQ0HW DQG 0 WXPRUV ZHUH DQDO\]HG IRU WKH
SUHVHQFH RI JDOHFWLQ LQ WKHLU OLYHUV SULRU WR WKH GHWHFWLRQ RI RYHUW PHWDVWDVHV 0LFH ZHUH
SHUIXVHG ZLWK VDOLQH SULRU WR KRPRJHQL]DWLRQ RI OLYHU WLVVXH DQG DQDO\VLV E\ ZHVWHUQ EORW &
/HIW,PPXQRVWDLQLQJRIOLYHUVIURPPLFHEHDULQJ0WXPRUVZLWKQRGHWHFWDEOHRYHUWPHWDVWDVHV
LQWKHLUOLYHUVIRUWKHSUHVHQFHRIPDFURSKDJHV)JUHHQDQGJDOHFWLQSLQN1XFOHLEOXH
DUHVWDLQHGZLWK+RHFKVW'DVKHGER[HVLQWKHWRSURZKLJKOLJKWWKHDUHDVKRZQLQWKHERWWRP
URZ 6FDOH EDUV LQ & DUH —P WRS DQG —P ERWWRP 5LJKW 4XDQWLILFDWLRQ RI JDOHFWLQ
SRV)SRVFHOOVLQWKHOLYHUVRIPLFHZLWKRXWWXPRUV7QRQ0HWWXPRUVRU0WXPRUV(UURUEDUV
DUH VHP PYDOXHV LQ & ZHUH GHWHUPLQHG E\ 2QHZD\ $129$ ZLWK 7XNH\¶V 0XOWLSOH
&RPSDULVRQ7HVWPP
)LJXUHTumor-derived soluble factors induce the mobilization of galectin-3+ myeloid cells into
peripheral circulation early in tumorigenesis:LOGW\SHPLFHRUPLFHEHDULQJ7QRQ0HW7RU
00WXPRUVZHUHDQDO\]HGIRUWKHSUHVHQFHRIJDOHFWLQOHXNRF\WHVLQWKHLUSHULSKHUDO
EORRG E\ IORZ F\WRPHWU\ $ $QDO\VLV RI VWDLQLQJ IRU JDOHFWLQ DQG &'E RQ DOO OHXNRF\WHV
IURPPLFHZLWKRXWWXPRUV177QRQ0HWWXPRUVRU0WXPRUV1XPEHUVUHSUHVHQWSHUFHQWDJHVRI
DOOOHXNRF\WHVWKDWZHUHGRXEOHSRVLWLYH%3HUFHQWDJHRIDOOOHXNRF\WHVWKDWZHUHJDOHFWLQ
& 3HUFHQWDJH RI DOO FHOOV WKDW ZHUH &'E ' 3HUFHQWDJH RI DOO FHOOV WKDW ZHUH
&'EJDOHFWLQ ( 3HUFHQWDJH RI DOO OHXNRF\WHV WKDW ZHUH *U ) 5DWLR RI &'E/\
&KL FHOOV WR &'E/\&OR FHOOV * *DOHFWLQ H[SUHVVLRQ RQ /\&OR DQG /\&KL FHOOV P
YDOXHVLQ%)ZHUHGHWHUPLQHGE\2QHZD\$129$ZLWK7XNH\¶V0XOWLSOH&RPSDULVRQ7HVW
PYDOXHVLQ*ZHUHGHWHUPLQHGE\7ZRZD\$129$ZLWK%RQIHUURQLSRVWWHVWP
PP
)LJXUH Secretion of IL-6 by tumors induces the rapid mobilization of CD11b+galectin-3+
leukocytes $ $QDO\VLV RI &'EJDOHFWLQ P\HORLG FHOO PRELOL]DWLRQ WR SHULSKHUDO EORRG
IROORZLQJ LQMHFWLRQV RI FRQWURO PHGLXP ZKLWH 0 OLQH FRQGLWLRQHG PHGLXP UHG RU
UHFRPELQDQW PXULQH JDOHFWLQ VXSSOHPHQWHG FRQWURO PHGLXP JUD\ % $QDO\VLV RI
&'EJDOHFWLQP\HORLGFHOOPRELOL]DWLRQIROORZLQJLQMHFWLRQVRIFRQGLWLRQHGPHGLDIURP0
OLQHVFRQWDLQLQJHLWKHUDFRQWUROKDLUSLQVK/XFEODFNRUJDOHFWLQKDLUSLQVUHG &/XPLQH[
F\WRNLQH OHYHOV RI WKH FRQGLWLRQHG PHGLXP ' *HQH H[SUHVVLRQ PLFURDUUD\ DQDO\VLV RI Il6
H[SUHVVLRQ LQ DOO FHOO OLQHV IURP WKH IRXU FODVVHV ( IL6 H[KLELWV D JDLQ RI FRS\ QXPEHU LQ
KXPDQOXQJDGHQRFDUFLQRPDVFRPSDUHGWRQRUPDOOXQJWLVVXHRUEORRGP îLQWKH
³/XQJ DGHQRFDUFLQRPD´ GDWD VHW DYDLODEOH IURP 7KH &DQFHU *HQRPH $WODV ZHEVLWH VHH
0HWKRGV)*$QDO\VLVRI&'EJDOHFWLQFHOOPRELOL]DWLRQWRSHULSKHUDOEORRGIROORZLQJ
LQMHFWLRQV RI FRQWURO PHGLXP RU PHGLXP VXSSOHPHQWHG ZLWK UHFRPELQDQW PXULQH ,/ +
0RELOL]DWLRQ RI &'E OHXNRF\WHV LQ ZLOGW\SH DQG JDOHFWLQ NQRFNRXW Lgals3-/- PLFH
IROORZLQJ,/LQMHFWLRQV(UURUEDUVLQ&DUHVHPµ1'¶QRWGHWHFWHGPYDOXHVLQ$DQG
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
+ZHUHFDOFXODWHGE\2QHZD\$129$ZLWK7XNH\¶V0XOWLSOH&RPSDULVRQ7HVWPYDOXHLQ
*GHWHUPLQHGE\6WXGHQW¶VWWHVWPµQV¶QRWVLJQLILFDQW
)LJXUH Elevated Thomsen-Friedenreich Antigen presentation promotes increased galectin-3
adhesion in metastatic populations $ 3RWHQWLDO FDUERK\GUDWH OLJDQGV IRU WKH JDOHFWLQ
FDUERK\GUDWHUHFRJQLWLRQGRPDLQ&5'7KH7$QWLJHQ*DOȕ*DO1DFĮ267LVVSHFLILF
IRUWKH&5'RIJDOHFWLQDQGN$FHW\OODFWRVDPLQH/DF1$FELQGVWKH&5'RIDOOJDOHFWLQV
DQG H[KLELWV LQFUHDVHG DIILQLWLHV ZKHQ LQ D SRO\PHULF IRUP 7KH $ % 7\SH EORRG JURXS
DQWLJHQVKDYHUHSRUWHGDIILQLWLHVIRUDYDULHW\RIJDOHFWLQVLQFOXGLQJJDOHFWLQE\JO\FDQDUUD\
DQDO\VLV % 2Q QRUPDO FHOOV ELQGLQJ RI JDOHFWLQ WR WKH 7$QWLJHQ RQ JO\FRSURWHLQV LV
RFFOXGHG DV WKH HSLWRSH LV PDVNHG E\ IXUWKHU JO\FDQ H[WHQVLRQ 2Q PDOLJQDQW FHOOV DEEHUDQW
H[SUHVVLRQ RI WKH GLVDFFKDULGH ZLWKRXW IXUWKHU JO\FRV\ODWLRQ SHUPLWV JDOHFWLQ ELQGLQJ &
3HDQXWDJJOXWLQLQ31$ODEHOLQJRIWKH7$QWLJHQRQUHSUHVHQWDWLYHFHOOOLQHVIURPWKH7QRQ0HW
EOXH 70HW JUHHQ DQG 0 UHG FODVVHV DV GHWHUPLQHG E\ IORZ F\WRPHWU\ ' %LQGLQJ RI
JDOHFWLQ IOXRURSKRUH FRQMXJDWHV LQ WKH SUHVHQFH RI D JO\FDQ FRPSHWLWRU IRU JDOHFWLQ N
DFHW\OODFWRVDPLQH /DF1$F RU FRQWURO GLVDFFKDULGH VXFURVH () +XPDQ 16&/& WLVVXH
PLFURDUUD\V ZHUH DQDO\]HG IRU VXUIDFH 7$QWLJHQ SUHVHQWDWLRQ E\ 31$ VWDLQLQJ ( 6DPSOH
WLVVXHVWDLQLQJLQOXQJDQGO\PSKQRGHWLVVXH([DPSOHVRIWLVVXHVVFRUHGDV31$QHJDQG31$SRV
DUH VKRZQ)4XDQWLILFDWLRQRI70$VSRWVIRU31$VWDLQLQJµ1¶QRQFDQFHURXVWLVVXHµ&¶
FDQFHUWLVVXH*31$ODEHOLQJRIKXPDQ16&/&FHOOOLQHVDVGHWHUPLQHGE\IORZF\WRPHWU\
PYDOXHV LQ ) GHWHUPLQHG E\ )LVKHU¶V ([DFW 7HVW 6FDOH EDUV LQ ( DUH —P —P IRU
LQVHWV
)LJXUH Metastatic cells downregulate *FQW and upregulate 6WJDOQDF $ /HFWLQ EORW RI
VXUIDFH SURWHLQV &HOO VXUIDFH SURWHLQV ZHUH LVRODWHG IURP UHSUHVHQWDWLYH 7QRQ0HW 7 70HW
7DQG00OLQHVDQGUXQRQ6'63$*(JHOV0HPEUDQHVZHUHEORWWHGIRUWKH7
$QWLJHQ ZLWK 31$ % *HQH H[SUHVVLRQ PLFURDUUD\ DQDO\VLV RI DOO JO\FRV\OWDQVIHUDVHV 7KH
DYHUDJHH[SUHVVLRQIRUDOOSULPDU\WXPRUGHULYHGOLQHVRUGLQDWHLVSORWWHGDJDLQVWWKHDYHUDJH
H[SUHVVLRQIRUDOOPHWDVWDWLFOLQHVDEVFLVVD6L]HRISRLQWVUHSUHVHQWVWKHDEVROXWHGLIIHUHQFHLQ
H[SUHVVLRQEHWZHHQWKHFORQDOO\UHODWHG70HWDQG0OLQHV7DQG0UHVSHFWLYHO\7KH
FRORU UHSUHVHQWV VWDWLVWLFDO VLJQLILFDQFH RI WKH GLIIHUHQFHV EHWZHHQ DQ\ WZR DGMDFHQW FHOO OLQH
FODVVHVLH7QRQ0HWYV70HW70HWYV11YV0DVGHWHUPLQHGE\6WXGHQW¶VWWHVW7KHGDVKHG
OLQH UHSUHVHQWV HTXLYDOHQW H[SUHVVLRQ EHWZHHQ WKH PHWDVWDWLF DQG SULPDU\ WXPRUGHULYHG OLQHV
& T573&5 DQDO\VLV RI WUDQVIHUDVH JHQH H[SUHVVLRQ 7RS JHQH H[SUHVVLRQ RI Gcnt3 DQG
St6galnac4 LQ UHSUHVHQWDWLYH 7QRQ0HW 70HW DQG 0 FHOO OLQHV %RWWRP &*Q7 Gcnt3 FDQ
WUDQVIHUD*OF1$FWRWKHFRUH*DO1$FRI7$QWLJHQE\DȕOLQNDJH6WJDOQDFFDQWUDQVIHUD
1HX$FWRWKHFRUH*DO1$FRIWKHVLDO\O7$QWLJHQ'GCNT3H[KLELWVDORVVRIFRS\QXPEHU
LQKXPDQOXQJDGHQRFDUFLQRPDVFRPSDUHGWRQRUPDOOXQJWLVVXHRUEORRGP îLQ
WKH³/XQJDGHQRFDUFLQRPD´GDWDVHWDYDLODEOHIURP7KH&DQFHU*HQRPH$WODVZHEVLWHP
YDOXHVLQ&ZHUHGHWHUPLQHGE\2QHZD\$129$ZLWK7XNH\¶V0XOWLSOH&RPSDULVRQ7HVW
PPP
)LJXUHKnockdown of 6WJDOQDF reduces galectin-3 binding and prevents metastasis LQYLYR
$$QDO\VLVRIJDOHFWLQELQGLQJWRWKH0OLQH0IROORZLQJNQRFNGRZQRISt6galnac4E\
UHWURYLUDO WUDQVGXFWLRQ RI VKRUW KDLUSLQV WDUJHWLQJ St6galnac4 RU D FRQWURO JHQH /XF ILUHIO\
OXFLIHUDVH E\ IORZ F\WRPHWU\ () :LOGW\SH PLFH ZHUH WUDQVSODQWHG ZLWK *)3 0 FHOOV
EHDULQJWKHVKRUWKDLUSLQWDUJHWLQJSt6galnac40VK6WJDOQDFRUFRQWUROKDLUSLQ0
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
VK/XF E\ LQWUDVSOHQLF LQMHFWLRQ 7ZR ZHHNV IROORZLQJ LPSODQWDWLRQ WKH OLYHUV RI PLFH ZHUH
H[FLVHG DQG LPDJHG % 5HSUHVHQWDWLYH OLYHUV DV YLVXDOL]HG E\ IOXRUHVFHQFH WRS EULJKWILHOG
PLGGOH DQG KHPDWR[\OLQ DQG HRVLQ VWDLQLQJ ERWWRP & 4XDQWLILFDWLRQ RI WKH QXPEHU RI
VXUIDFH QRGXOHV RQ WKH OLYHUV 6FDOH EDUV LQ % DUH PP PYDOXH LQ & ZDV GHWHUPLQHG E\
0DQQ:KLWQH\WHVWP
)LJXUHT-Antigen presentation promotes metastasis through interactions with galectins in the
metastatic niche. 6FKHPDWLF PRGHO RI JDOHFWLQJO\FDQ LQWHUDFWLRQV LQ OXQJ FDQFHU PHWDVWDVLV
3ULPDU\ OXQJ WXPRUV KDYH KLJK H[SUHVVLRQ RI EUDQFKLQJ JO\FRV\OWUDQVIHUDVHV VXFK DV &*Q7
Gcnt3) DQG ORZ H[SUHVVLRQ VLDO\OWUDQVIHUDVHV VXFK DV 6W*DO1$F,9 WKDW DFW WR SURPRWH O
JO\FDQEUDQFKLQJDQGHORQJDWLRQ%RWKWKHSULPDU\DQGPHWDVWDWLFWXPRUVVHFUHWHWXPRUGHULYHG
VROXEOH IDFWRUV VXFK DV ,/ ZKLFK DFW WR PRELOL]H &'EJDOHFWLQ P\HORLG FHOOV LQWR WKH
SHULSKHUDOEORRG*DOHFWLQOHYHOVEHFRPHHOHYDWHGLQWKHOLYHUHDUO\PHWDVWDWLFQLFKHZKHUHLWLV
SUHVHQWHG RQ PDFURSKDJHV 0HWDVWDWLF FHOOV DGKHUH WR JDOHFWLQ DQG WKURXJK LQFUHDVHG 7
$QWLJHQ SUHVHQWDWLRQ ZKLFK LV PHGLDWHG E\ DEHUUDQW JO\FRV\OWDQVIHUDVH DFWLYLW\ 'HFUHDVHG
H[SUHVVLRQ RI WKH EUDQFKLQJ WUDQVIHUDVHV DQG LQFUHDVHG H[SUHVVLRQ RI VLDO\OWUDQVIHUDVHV SUHYHQW
JO\FDQHORQJDWLRQDQGSURPRWHSUHVHUYDWLRQRIWKH7$QWLJHQZKLFKLQWXUQPHGLDWHVJDOHFWLQ
DGKHVLRQ
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.
Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Aberrant glycosylation promotes lung cancer metastasis
through adhesion to galectins in the metastatic niche
Nathan E Reticker-Flynn and Sangeeta N. Bhatia
Cancer Discovery Published OnlineFirst November 24, 2014.
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-13-0760
Access the most recent supplemental material at:
http://cancerdiscovery.aacrjournals.org/content/suppl/2014/11/22/2159-8290.CD-13-0760.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 [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer
Research.