DTIC ADA426079: Ubiquitin Pathway Enzymes: Coactivators of Nuclear Hormone Receptor and Their Role in the Development of Breast Cancer

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by  Defense Technical Information Center

an

rd Kunbar: DAMDLT-UC. 1.0:

Conchi vars of Muclesr
Role iz the Develogment of

Toxmone Receptor an
hese: Sazcer

PRINCIPAL CNUESTIGATOR: afar Nawax, Po

CONTIACTIKG ORGANIZATION: Baylor College of Vedi cine
hiopato-, "asaa 37033

REZORT DATE: Jaaver:

Nek GY ARPORT: Final

PURPARSD SOF: U.S. Army Medical Roscarch ard Materiel Cowrend

ext Delzick, Maryland 21722-5012

EXSTRIRUTION STATEMENT: Approved fox Fublic Release
Digseibation Unlinited

the views, opinions ana/oz findings con:aincd in thie reser: are
those of the « (2) aad should not be construed an ay offisial
naparonent of che Aeay go2-Lica, goLley er decision mless 59
dasignased by ovhox docunontasion

srsranuancor 20040907 015
REPORT DOCUMENTATION PAGE

Fon Approved,
OMe Ne. 674-0168

‘deo oki Senuary 2924 Final (25 gum 60 ~ 12 Dec 02)

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Table wf Contents:

Tatra

Body

2

Key Reseurch Accomplishments,

Reportable Outeanes.

Contlusions..

Reforewees.

Appendices...
Intreduetion

‘irous,esmuer isthe lending cause nf deulh in Annetican women. This antcipuled Usut ane wrt
‘out of zen will develop breast cancer a sotne poi during kere (Nicholson 1979 Nicholson et
al 1986; Nrssity 1994; Nichalsen et at. 1995; Parker el al. 1997; Marrs eat. 2001). Aliboughe
in recent years sigaiticane progeess has been made in detection ard teeatment of the disease,
uch of che moleculae bosis of the disease temains uiknown, This fact highlights the reed to
identify and wivderstarl the molecular basis assuciated with eas! cancer development antl
progressicm

‘Steroid hottnoues, esttogen and ploges‘erone, play important role in che develogment and
progression of beast cancer (Benner etal 1988: Clarke et al 1989; Clarke o al, 1992; Elles et
6, BUG), Kstrogeus and progesterones exert thelt biological effects on aryet Ginwues Unraugh
inuravetluiar roceptar proteins, the estrogen {ER) and piogestecone (PR) receptors (O'Malley
1990; Tsai and O'Malley 1994; Hatina and Reischig 20K). Thene revepiars contain cba
structural motifs which include a less well conserved canina-lertnal aclivatcm fiction (ATS)
fat effects transcription elfisiency, which hts the hormone indepeaclent activation tunction; a
ential DNA-binsline domain, which modinees receptor bind:ng to specific BNA enhances
sequences aud determine Greet xene specificity; smd» carboxy-terminal hacmone-binding
domain (UBD). The HED contuins activation function 2 (AF 2); the segion mediates die
hormone sopendent activation function of the rectory (O'Malley 1990; ‘Isai and © Malley
1904; Harina and Relachig 2KIO)

in order to activate gene tancsiptiom, THR und PR undorgo a seros of well defined steps. When
Tpounl Co hormone. those receptors undergo a eanfermatinasl change, slissoecation trom cellular
chaperenes, reveptor dimetization. phosphorslatian, interaction with corctivators and
sectuitownt of chromatin modifying cazyine activities such «i hisiome uocty] tewnstegnse activigy
(HAT) and ATPase activity, DNA-binding st an enkancer element uf the lunge: gene, and
aucbseqqtent roeraitment of basal wanacription factors to forma a stable pecinitiacion comix (PIC)
(Harwity et ab. 1996; MeKeuns er al. 1994; MeKecay 1999; Chen 2000). Those events are
Tultowel by up-c eoven-regalstion of saget gene expression,

Cosetivators represent a grossing class of proteins, which interact with reveptars in a gand=
specific manner and serve (0 enhance sheir ¢ranscriptional activity. Priv th their identtieation,
conctivators wets piodicted ww exis! based ape expsrimenes, hich showed thal. different
receploct vamnpels for 4 ftsitng pool of accessory fhetors required for eptimal wanscriptin.
Stimulation of one receptor resulted it teans-tepression of wnather Foceptor, indicating the
depletia of a coramon eoactivater pool Bkacquel el al. 19R; Meycr etal. 1989; Shemshedini et
al, 1992}. A nurnder of coactivators have been closed lo date, incloding SRC-1 (Onate et al
1998), HEE (GRIPA} (iioag et al. 1996; Voegel ct al. 1996; Hong et al. (997; Vooge! ot al
1998), pCTP (ACHIURACBAIBI/TRAM.D (Anvick e¢ a, 1997: Chen at al. 1997: Li at el
1997; Takeshilu otal, 1987; Torch etal, 1997), PGCs (Puigserver ct al, 1998), SRA (Lan els
1999), CEP (Tkanen et al, 1997; Aatnisalo et al. 1998; Fromsdid ct al, 1998), EG-associated
protein (EG-AP), and ul conjugating enzymes such as UbeH#SR, UhelT7 and Ube
(Now ct al 1995p: Poukka et al. 1999, Poutdn otal. 20003 and this Vist is yrtosing apie cry
Dy ay
Coaetivators wen: originally envisions Ip serve a beldging rote, Tnking the receptor to the basal
teanscription toachinery Pugh and Tjian 1992: Tjian and Manietis 1994), Recently, the
functional role of coactivators has expanded by the observation that they have bean shown 10
possess euzymatic activities that may cont bute to thelr ability te enhance receptar mediate
Iwansotiption: SRC-1, pAUG/CBP, anc ACTR (RACHAIB1) possess a histone acetyl uansfernse,
HAT, activity (Ogryvke et al. (996; Anzick el al. 199%; Li el ab. 1997; Spencer et al. 1997;
‘McKenna etal. 1998; Coilingwdsd eal. 1999: Chen 2000) and members of SWISNF complex
contain an ATPase activity (Dunaiet et al 1994; Muchacdt xt al. 1996; Wanye al. 1996; Reyes
fel ul, 1997). Tigundactivnted receptors ure thoagh! ly bring HAT und ATPase wctivilics
‘containing coactivators to the chrematia survounding the ceceptct, disrapting che Socal repressive
chromati, seuctue by aoetylating histones and possibly other elomatin associsted factors and
catalyzing the uncoupling of ionic ixteraccans between histones and their sufateate DNA
(Dunaief 2¢ al. 1994; Machardt et al. 16; Ogryvin et al. (994; Wang et al. 1996; Vang el
1996; Reyes et al, 1997; Spencer et al. 19971, Because of their abiiqy uo enhance reveptor
ancdinted gous expeession, coaetivauyis are Uavught us play an important role in regulating dhe
magnitude of the biological cesponses to ocaianes (Xi et al. 1998; MeKecna 1994; Leo and
‘Chen 2000; Xu e¢ al, 2000), "The level of coactivacar expcession is etitical in detecenining the
netiviey of the receptor m targse tisucs and variations in hormone responsiveness seen in the
populat:on may bs due to differences in coactivator levels

ic xevopted that couetivators sithcr possess ar bring IIAT and ATPase activities w the
promoter region of the target genes ind presomubly manifest par of ehcr in vivo cnactivatien
Juntions through these onzymutic activities (Dumaicf et a. 1994: Mnchard: cc at. 1998: Ozeyzka
Lal. 1996; Wuny ot al, 1996; Reyes ct al, 1BBT Spencer et al. 1997). Recent identification a
the exzyies of dhe ubiyuilinpratensime wl ubiquilin-ke petty as covctivators 3y Mry ONT
laboratary and ofhces added 4 nese List in Uhe enativator Tek, These stidce suggest thatthe
Ubiquitin-conjugeting enzymes, Ubel 5B, UbcEl7 and (hed end the F3 ubiuitin-proteinHigases,
BE-AP and REFLRSPS. imeract with members of Ure steroid hormone reeeptar superfamily
inclucirg BR and PX and wioduiste their vansactivedion function (Lulwof and MeDannell $996;
MeKenma e: ul. 1998; Nawaz ot xl. 1999; Poukka et al. 1999; Poukdka ef al. 2009). Similarly,
nother coactivaur pratsin, yeast SUGH, sn ATPase sabunit of the 268-proteasemte comple alia
interacts with ane mvidulates steroid lgxtmone ceceplas fonction ¢Hraser ct a, 1997: Makino al
1997; Musuyemia and MecDoralc 1998). Instead of FAT activity, this ereup of conctivutors
postexses other oneymatic activities such a8 ubiquitin conjugation, ubiquitin ligation and
rrolease activities, However, a common theme hecieen the (wn gnoups of iacivals i Usa
both peskess wore sort of cuzymaticsetiviy

As mentioned sbove, ey laboratory has ienrifid ubiquisia pathway enzymes a8 conctivaters of
the nnciear hormone receptor supeclumily. We have cloned sn ES ubiquitin-protein ligase, E-
AP s steroid hormone receptor interacting proteis uring u yous! to-hy'arid screening ay, BS
AP eullanves die hormone-dlepuncent transcriptional activity of stcrokd hormone secaprors, PR,
ER. andiogen (AR) and glucocortigoid receptors (GR) GNawaz of al, 19998). BG-AP” was
prcviously identified asa protein of 100 kDu, present bors in the cytepesm wid the nucleus, EG
AP medinces che interaction of buen papillomavizuses type 16 anul 18 BF proteins with p33, 8
[provth-suppressive and tumer-suppressive protcin, The BS/EG-AP camplen specilically
interacts with p52 sel promotes the dsgratfuian of p33 ia the ubiqu‘tin-proteaseme prowein
degnadation pathooy: Huibregta tal 1591; Fchnegise etal. £993). AS mentioned above. E6-
AP is a member of he TS class of functicmally related ubiquitin-protein Uigases. #3 enzymes
Ihave boon proposed to play u rmjir role in defining substrace specificity of che ubiquitin syscem
effncr etal. 1993; Huihmeise et o. 199%b: Hluibregts® 2 al. 199Sa), Protein ubiqoit nation
alsa involves wo aller classes nf enzymes, namely the FL ubiquitin activating eavgine CUBA)
and many E2 ubiquitin conjugating enzymes (UBCs). ‘The UBA fist activates ubiquitin in an
ATP-dependent manner, “The activated ubiquitin dion forms a thioester bond between the
carboxyl-tenmine! glycine rosiduc of ubiquitin and w cysteine residue of the LAA. Next.
ubiquitin :s trmaferrod fiom the El ro one of the soveral E2s (UBCs). prescrving the high-onerzy
thigestor bomiL Ta wsme vases, ubiquitin ik transferred Prectly fram the T? ta the target protein
throug un isopeptiie band bereen the eaino group of Tysine resus OF the Target proicin
and the earboxyl-termious at ubiquitin, In other Instances, the danster of ubiqeitia from UBC:
to target proteins proceeds through an 3 ubiquitin-protcie ligase imermediate such as E6-AP
sCicchrnover 1982; Ciechanover and Schovartz 1994),

‘The F2 ubiquitin coajogating enzymes of the ubiquitin zathwway, ObcHSB end UbeH7 (LECS)
als act an ceuetivators of steroic honmone receptors. Furthermore, we have also demonstated
that the FR provein, which is a major miodulster of nerenal mammary gland development and
Dreast Lume developauent, is rapidly degrade im manamalion cells in an estrogen-depeadorc
manner, Treatment of mammalian cell. wilh the proteesome inhibitor, MG132, which
specifically blocks the protease activity of the proteasome, blocks PR degradution, This suggests
that HR protein is degraded divmugh dhe ubiquitis-proeauorce pathory (Nawas el al 19996), In
snblition, oar results also suggest that the esttogea-dependent degradation af TR comelates with
hhormune-lepeniion; BR activation becamse MGI32 not only blocks BR protein deyradarian hut
als Mack its ativation. Our i vite studios suggest that ER degradation chve-ved in
pburesnotian cells is dependent on the UBCs. UbcHSH and Ube anid ubiquitin-pruteascme
pathway [Naver et a. 1999). ‘These observations raise the question as tn why ubiquitin
ppathovsy enzymes. and ubiqucie-dependent protein cogrsdation ate linked to stenvid hormone
roveptot activation. Conxidering tha: the mimseriptionnlly serivo reeptor is associated wth a
verse group of proteins und firms « proinifistion complex. it is passibte that sabsequent lo
receptor detivation of teanscr ption, ubiquitin madiatotl degradation of the receptor way be a
rcchanism witeh dissociates che prenitianion virmplex. Teil be nccessary te dissoeiare the
pécinitiation complex chrough targeted protein degradation since the synergistic incractions af
rmhiple gunscription factors may make passive dissaciution wf borr:one and conctivatars
ianpossible ur fine consuming. Addlicians(ly. it Is possible dhat hormone-ieduced receptor
dogradation serves to curtnal physiological responses to scroid biemones ullinudely Ting he
expression af steroid-eesponsive genes

1 bas been shown that altered expiessiin OC uae nucleas receplr casesivator, ATHY, commits
‘the development of hormone desendent beeast and ovurian tances. Interaction of ATE. SRC-
1, TIF2, and p/CTP with CREF p3D0 is important for the coactivation funclioe. The,
overexpression oF lost of expressien of any af these conetivators could potonally pertib sient
integration by CBP! p300 aad affect mulliple transduction pathways (Anzick ct al. 1997),
Recenly, it has also beeu shown diet another stervid reveplor couctivator, SKA, 15 as clovated
cio Lamors (Murphy et al. 2000). Purthermore, we have alka shown that F6-AP iy
pverexprensed 25-45 fold in 90-95% of tumors using a mouse ramnmery model of mullitage
lumorigenesis, EG-AP is overexpressed only in tumors but not in the intermeil ate steps of
tumorigenesis (Sivaraman ct a. 2000),

“The purpose of th’s research is to oxplore the possibility thatthe altered expression of UbcHSB,
Ubel 17 and E6-AP may conttibute to the develapmiont of breast carer. fn the cviginal propesel,
wwe proposed te explore this possibility by stuey:ng che expression profile gf UbcH4R, UncH7,
nine ER in vavious breast eaecer sell Fines ani breast cum bingy saraples, We alan piped 15
create novel in vitro models in stable cell lines, which will overexpress cowctivalor proteins,
“UbcHIST aud UbcH?.. We hove examined the expression patterus of E6-AP, UbcH?, with that of
[ER in various human breast conver cell linen und breast tamer inp samples. Adaiticsally, we
have also coselated the expression protile of EO-AP and UbeL? with thar of ER in breast taco
biopsies. Unfornmately, due to the Inek of good UbcHSB autibody. we ate enable to study the
‘expression profile of UbcHB, Lastead of UbcHSB, we bave examined the expression pretlle of
FE-AP in several Rroust cancer cell Les snd breast biopsy tumors, In erder to study the
expression prfie nf R6-AP andl UbsH? in human breast tumors, we have. examined 100
Ailferen breast cancer hinpsy sarnples forthe expression profile af TAP, ThcH7 und TR. Wee
ound an inverse cosrelatina between the exprasnion al T5-AP and the exprensim of TR im these
tumors, Tho Spearuian eauk Coelation Coefficient is 0.38 and dhe p value is (MH, indicating
thas this correlation 1s statistically sigtiticaut. ‘These data suggest a possible cols of EO-AP In
monmury gland development sme! tumorigenssis, However, We aid not find any statistically
significant earretation between the expression provis of Ubell aml ER in these turasr samples
Additionally. we have also eaniined the expression profile af TAP, UheH? ancl TR in extly
fand intermediate stage tumors. We found that there is na conrelution between the expxsion
profile of H6-AP and ER and UbeH? aud ER in early and intermedisie stage lwnors, The
expression of BG-AP is down in 30% of invasive breast carice:s ancl exprensinn oof F6eAP is
inversely someksted with ER .n anvasive broast cencers. Parthermme, we fod that TEA-AP:
selulous the exyrossion levels of ER both in vitro and i vivo, Another goal a this project is us
ereate novel in vitza models jn sts cell Lines, which will evecexaress coactivator proteins, E6=
AP and UboH?, In order 1 achieve this goal, we have constructed eh expression vootors for
stable cell uss, Our data suggest chat these vectors pradace bialogieally functional cacctivator
proteins, T6-AP andl UbcH7, Howcve:, we failed ro ovelexpcess either ES-AP or UheH? in
Sable cell Tne hath hy eonstitusve expression system and mateble expression system,

Body

su this original proposal, we hyyprthesivel thal ubiquifin-conjugsting onaynies, UbcHSH, UbcHT
and an FS ubiquitin-protein ligase, Bo-AP, are ioxgortant anadu‘alons af the steroid ho-mans
reteplor-medintel igral traduction pathway. cell araweh, aad val] oycle camino in te conte!
fof brecsi cancer development. Yn onder to test this hypothesis we propose following objectives:

+ Expression analysis of eudogenous ubiquitin-coujugutiag enzynus, UbeTIST und
UbelI7, und ER in breast eanoer cell lines and human breast tumor hlopsies. Th
compore the espression patterns of UbeHSB and UbcH? with that of EM.

+ Design and development of stable in vitre modcls of UheHSB and UbeH7
‘overexpression in the breast cancer cell
es al stably transfected cell Tines that overexpress
ity of these stably transfected

+ Analysis af the grawth propert
UbelI3B and UhelIT and io vivo analysis of tumorigeni
well Fins in athymic nude nulee.

resslon analysis of endogenous ubiquitin-conjugating enzymes, UbcHSB and UbeH7,
and El im breast eancer cell lines and huruan breast tumor biopsies. ‘Then compare the
expression palerns of UbcHST nd UbeTT7 with that of ER,

(One ofthe sims uf this pruposa is to rest the capression of endogenous UbcHSE, UbcH? and ER
jn hunmwm breast cancer cell Hines aad human breast cumnor biopsies. ‘Then compare tac
expression profile nf Uhef 150) andl WbeTT? with that of TR. We: have examined expression levels
pF UbelI?. E6-AP. and ER i HO" different hrecst unos and expression of p53 in 20 different
tums. Additicnall we have alse examine Ihe expression profile of UbcH?, E6-AP snd ER in
different breast cancer cell Hees. Dug ta the lick of the avuilabilty af tho UbcHSB antibody, we
fare unable 10 examine the expression profile of UbeHSR. Furthermore, wo were not suceossfol
iu generating a good UbeITSB antivady. Since there protsins sre ulsy to-gote of tho ubiquitin-
proteasome patboray, we did not analyze the mRNA levels of UbcHSH. Acklivionslly, UbcBSE
and UbcH? both act as £2 ubiquide-cogjugating enagmes for CA-AP, therefore, swe alia
examined Uhe expression nf F6-AP in ei ferent bxeast cance cell lass and bzeast tumor biops!
‘We round an inverse coreelatinn hetoween the expression of F6-AP and the expression af ER in
Jmman biopsy tninor samples, and we did mot find any statistically significant conelarioa
‘boteicen the exprossion profile of Ube 7 and TR,

‘Task Le Expression analysis of bs BR and EG AP in differer¢ breast cancer cell Fines,

analyved Ihe expression profile of UbsH, HR cud ES-AP in diferent breast concer cell

as MCP-7, T4-D, ZRIS-1 and MDA-MB.231, As a coctiil we have ass exsannined
the expression pratile of UbcH?, ER and EG-AP in HeLa (@ bers ice] earcienn cull Ting) cells
As shown in Figure 1, Hele call: expross high lovols of Ubel{7 protein cwvagrare tn th of
Aliffereat breast cancer cell lines (MCT-7, T47-D, 7R75-1 and MDA-MB-231). Furtherinore, in
Hefa cells, the UbcH? expression is boul zytsplusmie ard auclear, ‘Ihe exptessioa level af
Unc] in MCK-7, T47.D, ZR75-1 and MDA-MB-231 is moderate compare to that of HeLa
cclls. In T47-D cells the expression of UbeH? is orally nucleas whereas ia other breast ease
cell tines, MCE-7, 2R75-1 and MDA-MB-231, weak cytoplasmic stainiag af UhcHT ous
dobsorved, i addition 0 nutes: Sljning.

B. BR expression
Since we want ta compare the expression profi’e ot Ube(17 with that of FR, we also amalyecd the
expression of ER-alpha in MCh-f, L47-D, ZR75 1 and MDA-MU-231 eell Times, Tn this ease
HoLa cel line was used a a nevative ermteol. As shown in Figure 2. HeLs calls ate negekive for
ER expmesaicm. Similarly, i eho breast cancer cal line, MDA-MT-231, the FH expression was
lundetectsble. However this eel ing exsecssos UBCH? at maderate level. Tw vortras tr MDA.
MB-231, the MCP-7, T47-D and ZR7S-1 lines express both UbeH? and BR-alpla, The FR
‘expression is uuctear ix Giese ceflTnes
C. 66. 8P exprencion

Since UboTI7 act as an B2 ubiquitin-conjngating oazymo for F6-AP unl both the Ube]3? anc E6-
AP act as coactivators for ER, wo eocidetl tp analyze the expression peofle of BG-AP in breast
‘cancer ec lines. As shovin in Figure 3, the hrewst cancer exll Lines, MCF-T, T47-D, ZR75-1 and
‘MDA.MB.231 express high levels of ES-AP, The T6-AP expression is bath cytoplasmic and,
nuclear in MCF-T, 775-1 and MDA-MB-231 cat! lines. ‘The MDA-MB 231 cell line expresses
rioce F6-AP in nucleus thar in the cytoplasm, Similar 10 Ubcfl7, the expression of B6-AP in
‘TATTy cells is punely nuclear, To Mis case Tel a cel’n were used af a positive contiol far GAP
expression.

‘Task 2. [fect pfsternids on the expression of UbeTI? and B6- AP.

1 is possible Ghal_sternidhonmonet (estngenciprngestenines) may regulate endogenous
expression of UbeII7 and E6-AP in breast catcer call lines. To test this gossibiliy, MCE-7, a
Tarinone-deperatent breast cancer cell Tine was grown in Uhe megiun cnntaining stripped secusn
fora week. Afterward, cells wee grown either ia ths abseace or presence of steroid heremones
tor 4% hour: and the exprcssion pavtems of UbeHT and E6-AP weie deterred by Huorescent
immurocytochersistey. As shown in Figure 4, in MCE-7 cells, estrogen treatment has ae
significant effoot on te expression profile of LbcH7. ‘The UbsH? oxpression is identicsl both in
the absouce and presenec of estradiol. Similatty, progesterone Ueatment also has no signiticant
cifect on the cxprossion levels or UboH{? in MCF-T cells Gata not shown), Next we ask whether
eroids peatment xs any effect on the expression lovels of UbcH? in T47-D celts. As shown in
Tigure 5, esirogen trtment has no cffect or. the expression levels of Ube li? in ''47-D colls. the
expression levels of UbcH7 are identical oth in hormone trosted and ontrested colls, he seme
js uve for progestorone (data not shown). These data sugeest thatthe exyression af UbcHTT is ne
uniter ¢he control of staid hermaones,

Neat ve ask whether stercids egulile the expression WfEA-AP, ‘To test the sffect of estrogen on
tho cxpression pattern of EG-AP. MCE-7 cells were grawn in the mediurs containing sttipged
scrum for swwosk, ‘ten, cells were treated with either estrogen or vebice for 4% hours ard the
expression patter of ES-AP was determined by fhiotescent imrauneeytochemisicy. Figure 4
suggests tna the estrogen treatment havc no siguificane effect on dhe expression of EG-AP. Toe
EG-AP expression levels re identical holh in the provonce and absence of bomen. ‘This data
‘sugges¢s chat ES-AP regulation i wot under the camtral af sien,

As a conttl for these experiments, we alse onalyzed Ihe ofTeet of estrogens on the exzession of
PR anc HR, rhs boon cstablisted tha: estrogen upeegulate the espressian of PR proicin und it
down regulate the Ievels of ER in MCE-7 cells (Lonant et al. 2000). Aw expecta,
dernonsiras Chat estrogen trees inckcases the expicasion of PR prevein, In cootrasl, eno
down eegolates ER expression

‘Tak 3, Eopression anal

is of ER-alph

hg 17 ani TS. AP in breost tumor songs

As mentioned shove, the ubiquitin pathway enzymes, UoelI7 and E6-AP act as ccantivcars of
steroid hormone receplers. Purthermaane, we have sso demonstrated that eho BR protcin, which
18 « major modulator of normal mummury gland development and yeasc wena development,
Cepidly degrades! in-mummatiun cells in an estedgen-degendent ranger via the ubiquitin.
rotessorue pathway. Addicianally. ous in vitra seudies suggest that ER dogradat.on obscrved in
ranwmalian cells is dependent ou the UbeHT and ubiquirit proteasome pathway (Nawaz ctxt
19999). To explore the possibility thar the altered expression of UbcH? and £6-AP may
cantrnute tthe development of breast cancer, we analyzed the expiessioe profile uf UbcH7,
EG-AP and li in 100 advanced stage breast tamtor biops¥ samples by Western blot analysis and
:mununokistochemistry, Figure 8 aud 12 show tho expression protile of ERalpha in 56 different
‘aurnan comor semplcs. 23 (41%) ont of 36 brenst tumor samples expless significant amount of
-Ek-alpha, loowever, 33 (G94) camots express no oF degraded forms of UR prover. Since we
want (» corluts the expression profile of ES-AP and LibcH/ sith that of HR in breast mner
biopsies, se examined the expression profils of UbeH? nnd P4-AP in these tumers, igure 9
and 12 show the expression profile of UbcH? in human broast tmior samples, AS shown in
Figure 9, mujorty of the lumars expresses UbvH7, Only 213) tumors aro negative for UbeH?
Furthermore, we did not fiaé any statistically significunl correlation between the cxpression
profite of UbcH and ER in these cunesr samples

Since UbeH? sets as on E2 ubiquitia-conjugatinys enzyme fer T6-AP, we alsa examined the
expression profile of K6-AP in these unian breast cimoe samples. ‘To study the expression
rofile of B6-AP in beman breast tumors ve pertoroned Western Wot analysis usiay na E6-AP
specific antibody, Figuec TU shows the expression of FS-AP in 56 diferent tuna: samples, Toe
majority (S25 of the tumors expresses ES-AP. Turthermore, we found an inverse correlation
belseer the expression of hG-AP and the expression of BR iu diese tum. ‘The Spearmi Ran’
Correlation Cocfficiout is 0.38 and the p value is OHM, incicaliny Uhat this comclation is
sutistiewTy significant (Higuro 12),

It bas heen demonstrated that EG-AP promotes the degradation of pS vin che ubiquitin
degradation pathway. In the brain of L6-AP kavckout animuts, the protsin levels of p§3
accumilale earapered ro those of normal Ustermates. Therefore, we also analyzed the
endogenous expeessiva: of 53 provein from hrcast tumor Blapsies. «As shown in Migure 11, p$
sxpression was not detectable in most tumors execpt tmmor number 7 10, 13 and
Funhermote, hore was no stetistical correlation between the exprossion protlle of E6-AP and
1953. Presently, we are analyzing move eumor saniples for p5$ expression

‘Since we found an inverve carrelalim hotwoen the expression profile of ER-alpha and FS-AB,
‘we analyzed whethe: E6-AP and BR expression colocaliged in brsast cancer cell lies. Tn omer
to study the co‘ocalizatian a ER ant F6-AP, we pofarmed fluorescent imaxsnncytalieistry
on T7-D cells. As shown Hignte 13, the expressioc ol F6-AP eolesolized wita thar of EM (Gee
aictge figure). ‘Thess data soggest a it is possible that in vive EA-AP may promote the
degradation of BR throwgt: the vbiquitin-proteasarne pathosay

‘We have also examined the expression profile sf T6-AP in normal buraaa mammary tissnes by
imrunohistachemisoy using antl-E6-AP antibody. Fig. 14 suggests thor P6-AP iy highly
‘expressed in normal human mammary duets and almost every epithelat eell express EGAP
protein (iy. 14),