Table Of Content

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VIVLE: Biomarkers of Selenium Chemoprevenzion of Hroetate Cancer FA(RC.2KG TRESTISATOR: Yan Dong, Pi ORGAN 2AT-GM; Health Reaaarch Tneomparsted Buffale, NY 24262 IRON TATE: January 7604 Command EISYRBULLON STATENSN’ Approved for Publiz Release; Tiscrioution Ualimited ‘he views, opizions azé/or findings contained in this repo= those of the avther(e) and should not be construed os en ofS cia’ Bapavsnent. of the Army positioy, policy o- secision unless Gesiguated by other docunanzasio: 20040602 046 REPORT owe ho Ore 0158 DOCUMENTATION PAGE tence Heat Banusey 2002 Baeual Gusavy!1 gan 2003 - 31 ose 39¢31 on of praszaze Sancor | panor7-02~ | picuavtevs of selestum chonvoveve: i van bong, Pb. ‘erorT NUE Fen’ th Runmarch Tacoeporabed before, Mat EM: yan. dongososse] Iparit. org SScecv maRzil AND ADDRESSES) ‘AGENCY REPORT MOSER U.8. gray Medien] Keacareh ond Macerin: Command [POETRY HEE SEE fete approved for Bvblic kicleoec; Distribution Unlimited “Ths poe uty evanod dh ella ard clos ees of ete i he an-ogennagursive LNCaP eels Pyologal oacenirnion of seleniun produced u dove an espe ahamster at Gy pane ms tiescd 20, A nid docene oth uanscip ard poten lev! of wnrogen repr (AR) ar passe aig 8 tod ay carly ve 6h aggestirg thi the ile nance of uoyee & avaag Dy selenium iz nor 3eanacquence of oR nest ‘natin sxpeviannts shaved that seine os capac of acppreesre tb DNA bialng ese seoeeaeloting els of AR. (Gene ecpsision pring ors cored et one To ikaufy lor AR. ge’ cxpossivo 9 soln Wedulstion The eels ‘loved that selcoars war abe to ezunertund the expreston ofa subrt of 1z ional NKorea yorox. Thee yonsv aca ‘gh af in ht pons Ava flowrate dave overgnion, 2 0 found that slit fey alee be expres ‘oF (2 puptiveoorogence, zaoceupereenn yor, or genes inplcted en ne fers in aloe edapeaicae By cate ‘ekeant fenton fon fhe rcmartay da. hese thos unensced a misber of potnslly exci cles cept ee actin of ‘hulu in reetne ener prevention, Sele ur-rergreyive biorerkert, micresecay analysis, Wectanien of a Setooin coomaprevention Veedage=tred Unclave! tied Unesescities iintimices HS SITES eae Bee Cover... SF 298, Table of Contents. Introduction... Body... Kay Rasaarch Accomplishments. Reportable Outcomes. Conclusions, References. Appandices.. Table of Contents A major youl vf this project is @ identify suitzble biomackers af seleniur: chemoprevention in tuman prastate cell mode's, During this second yotr of funding period, we investigated tke eatlular and malecular changes rnatiated by selenium in the NCEP human prostate cancer cell Tine, Please see the allached article (Dong ef at Cameer Res, 64, 19-22, 2004) ural che manuscript (submitted t Cancer Res) in Appentis fir detailed description of the specilie aspects ol de tesearah, In the ariginal Staternent of Work, we proposed to look at the LNCaP prostate cancer cell Tine in the Fist year and the PC-3 cefl line in the second yest. ‘The reasor. taat we switched the souotce of tho work is that PC eolls aro relatively casy to tandle. Wo would like to ‘work out the experimenta! conditious in BC: 3 before we procesd te the LNCal cell Lin. We also proposed to pscform Affencirix GenoChip alysis in normal prostile cells in ‘order to identify genc expression changes induced by sc'onium. Bascd on our eaperisnce wilh the PC-3 cell ins, we found that the GoneChip analysis would goncrale volemingus aroun of dat, The challenge is he (6 extract oul etal information. Therefore, we developed a bivinlarmatic-hased aypaach ts Cather analyee uue GeneChip data, av detciled in the following ceotion. We would idke to postpone the sncey La novmal arastate cells until te bioiedtumaties-based approach is validated Detailed Description of the Hromoter-ased Mierosevay Data Mining Appronet In colfaharation with Ds. Tsiten Zhang, a bivirfirmalint al our Insitate, we developed a novel bioinformaics-based approach to fuether analyze the Allymeteix GensChip data gencrated in thc LC-3 cells. The ultimate goal is to characterize key ‘nnasctiption factors thet might mediate soloniurn-induced geste expression chacgcs. ‘Tbe underlying. premise of ihis xppronch is based on twa findametal principles: # Genes that are coordinately regulated should share camman regulatory elements en their Promoter regions. By identi(tng sueh common regulatory elements for a cluster of carly seleniuoresponsive genes. we should be able to deduce ihe trunseription factors ‘associated with these elements, TMese manseription factors are porential proximal largets of selenium, ‘Yo grons genes based on tht expression profiles accoss multiple rime poims, we wsed the Self Crgacicing Map (SOM) algoritian to do the analysis on all the genes that ure significantly moviulnel by MSA. ‘This acts nf malysis precocsd 16 distin alustors (hig. 1), We decided to welect ehesler 4 uv pursue the bioinformatics inlerogaon as an exemplary ease study. The characteristics ol'this cluster and the reavons for itt welention sre dolincated 2clow. F There are 372 genes in the cluslar Ihe sizable numbsr should be scfficicet to validate the easibiity and rel:ahitity of our higin ormaties apprich, The goncs in chis eiester ate all significantly downregnateé (based on staiaieal computation) by MSA ou'y af the 3-hr ts point, The entire efnstsr comuns co contr fevel of expressian atthe subsequent rime points of smalysis (Le, 6, 12, 24, 36 and 48 he}, This pactemn is consistent with # Cost mechani of weleniue teontrol a the setivites of wacscription factors. 4 Lae fast that these 372 genes are dovsnregnlated upon exposure ¢o selenium iiplics the they ure espressed constitutively. ts amy’ dita, the chunge in the ‘exgression ol'a gece that is constitmtively expressed in the eoowol sarcples is inherently’ more saliable compared fo the change observed with a gene raat is not ‘untmally expressed inthe cootro! samp. rece Pee a ; ans letriis. bcett (pitewss Serta st free: gam freed Pol of Change “Tle Paine de) Fig.l, Average gene esprian praie fe each SOM euser, The Loet- ‘rauatord dof shone far wu yan w ust nasa eraged and feted age daraton of teatnvce. Dat wre prevented > ment So. be printer ee anor tc o relrieve and analy the peomwster sequences af 287 genes (out of 372) wilh the ensioon Perl progean's. ‘The accessien umaaber of each transcript ws used to mip the transcrip to a Unigene clustsr andro absait: the Locus ID of Ure earrespoding gene. he Locus ID was then used to query the NCBI TawusT ink database in atder to fetieve the anique cfovence sequenes RefRea) cf The gene. The later was then melted against the ham yonnme assersbly a: the Colvessity of Califomia at Santa Cnwz using he GaldenPat Genome Browser to obrain its mapping information. Based 1 this Information, 1 kb of promoter sequence was relieved [ar euch yew. The anseiiption frctor-binding matifs presented in eack roraoter were chen profiled usirg the Match IPryrem und he TRANSEAC 64 tatsatiption factor database, To contol lr backgro:wid noise, cach of the promoter sequence was scrambled i geaeeate a rendom soqionce of fe sume hase cimpcsilion. The scrarabled sequence was then profited for (ranscriplion fuclor-biading motifs. ‘The process was repeated 10 times, and the average oceurence thequency for aaca binding motif was calculated and #ssigmed ss buchen “the binding mets with cigaiticantly higher frequencies (* 2 standard deviations} thaa, he backg-ourl noises were tabulated, fhe collective >inding-moti? profiles for a: the fpnmaoters were ther mcalyzed 10 soerch Tar common stanscription factor-binding elements, and the eamespinding wansceiption frets were elaseifial a» plentiat melden of selenium accion, Table J. Common ansceiption Pastor fn onder to confirm thal OKI is. powsntially a modiator of seleniur. uclion, we perlormed EMSA to assess fhe effer: of MSA on the DNA-binding activity of GKLE, [Nuslear entruois were prepared from °C 3 estls incubatz in the dasence or presence of 10 IMMSA for3,6.¢r |S. A 2h eat a GEL consensus elemenl, 5'-ATGCAGGAGAAAGAA * GGCGTAGTATCTACTAG-7", wes téed a8 4 probe in she EMSA. As shown in Fig. 2 MSA Ircalment resulled in an ok ixhuction of GRTF DNA-binding asivity. The specificity of (he interaction smd ue presence of GICLF in the DNA- pprein comptex weee confirmed by tho compottion eiperiment using an excess of un‘aboled GELF clement as ‘well as the supershift assay using an auvibadly ausins! GKTR, respectively (hig, 2). a “ (our mievoaetay data showed thar MSA uprogulates Ihe lovol of GEEF transcript sional ia our POA ell, We therefove ‘ana veouduetad Nowthern blot aualysis to caraborste. this observation As shawn in Fig, 3A, the incezase Fi. ef GLI mRNA lovel ocgurs ut early 0s 1 br post MSA trsaumenl. GKLF expression ‘was also exatinell in LNCaP cells. [ae changes wore very villa to that seeu in PC-3 calls (Fig, 32), Key Findings: Seleniumsinduced grew inhibition fe TNCHP eels is achieved niainly by Gr’G tell eycle atest couplod to an iulustion of apoptosis. Selenium significantly downresulates the expression of prostato-specific emtiven (SA) transcrip and prulein, and the decreases in androgen revepior (AR) transcript and protein (ollew a similar dose and time response pettern upon expoure to selenium, ‘Ie toduciion of AR and PSA expression ovcurs weil before any significant change in cell number 1% Scleuiuun inhibits the DNA-binding aud the ers-alivaling activity oF AR, 1% Mierowmray analysis shows that sleatara is able to countermand the expression of sasubsel ul 12 additional AR-togulsted genes. These genes cortain the androgen, responsive clemeat mol in the'r promovens The inforration suggests thal selenium might he uble ty dieety modclating tho primay targets a” AR sigrating. v The anay data also irdicales that seteniunt is capable of fvormbly allering the expression a! 12 putative peostare oncogenes, mmor-RRpressAT Yenes, oF wenes ‘implicated inthe tansformacion to mudrogen-independenzy, y Abioinforrnatice-basod array data ining apsroach was developed. ¥ GELF way identi as a potential proximal tergct of setonium. Reportable Qutcom: » Publication aud Manuscript: Tony. Tee, $.0,, Zhang, H., Marshall, J. Gao, A., and Ip, C. (2004) Prostate specific antigen (PSA) cxprcssion is dowr-regulaied hy” selenium through dismuption of audragen recep sgmaling, Cancer Res, 64, 29-22 Dons, ¥., Zhang, H.. Marshall, 1, Nowsk, K., Gav, AC. and Ip, C. (2004) Seleninim eoantermenls Lie exprasion of andzogen-ogulared gence and echer ‘targets implicated in prostate carcinogenesis. Cmncer Res, submited. > Abstracts and Presentations Dong. ¥.. Zhong, H., Nowok, NJ Hewthurn, T., and Ip, C. (2003) A Bioinfoumatics Approach to Delineute Seleniure-raediated Transcriprien Contec] of Gone Expression Changin. Priceedinge of the dmetican Association far Cancer Reset, 44 (2 Raliony: 774 Dong ¥. Zhang, H., Nowak, NJ. Sxsthorn, T., an Ip, C. (2003) 4 Bioinformatics Approich la only the Molecular Mechanism of Seleuiun- sodiated Gene Expression Changes. Poster resented atthe Bdseard A. Smauckler Memoris! Pathabinlogy of Cirwar Wurkstup, Keystoce, Colorado. Dong. ., Zhang, 11 and Ip, C. (2003) Selenium aud genomics: a pas de deux uo ccauco: contol, Poster proecntsd at the "© Armual Intometismal Conlerence on ‘vonfiers in Cancer Broventign Research, Phoen'x, AZ. Dang. ¥., Zhang, 1, Nowak, NJ, Marshall, .R., ad Jp, C. (2003) Sofonium ‘Affects an Auray of Androgen Regulated Canes a-l Other Targets Implicated in Prostate Carcizogenesis: Evidence ftom cDNA Micrinrray Analysis. Poster prosented atthe Prostate Cansor Foundation 10! Annual Scientific Relrezt, New York. KY. & Funiting received: DOD New tnvestigstor Avond (PI) ons 1907 © Title: GRUP asa Novel Target in Selenion Chemopreventior of ostate Cuwer “ Aveard por year S75,0K 1% Bmplayment applied for: Assist Member, Dopl. of Cancer Chemuprevention, Roswell Purk Causer Trslitule, TutTula, SY 14263 Avancus in Brief APPENDIX 1 Prostate Specific Antigen Expression Is Down-Regulated by Selenium theaugh Disruption of Androgen Receptor Signaling ‘Yan Dawg Soo Ok Leg? Haltne Zhang? Jnmes Marshll? Alla C, Gan Fei sent af pratt stgen Apne nd [anager tno nd ran ene or’ freer pan erat at gaa ene bag se eel en aston oot wg ctf tent ee he Ua ‘Snes rey ear ne ee 1S cnt ond 2 deme omprve ere (Th ge ie ages se ine Tn freon inthe Sh Bain hs rn a pn a gu A arrrnive way thi can ates fea th Clie armen of ct ane we Re wal sll Br faring the pat nF i Wigley ast n'a pec ‘Bedoaas,lsso ean es: poveron ri isa fie ener escarole nsoordny en per S24 oe 12 bj ne rok re eh egremneion with niin ne stein ws 2nd iy aloe sate so dese Ly ‘Spa Reso ands Oy Dango) soe hac alec Iaeé Lumen poses coos ee ew, Woche ce pe bbiche olen tation py on ede! prozac ll rc aoe eee vies PSA) pele he rl 2fenragenrecar(AR} we aa he fer de dagen und pres ol Fava ena. vow of Ur ‘asleanntion sf stlsbonesol Siege cane SRR tient Peyton ese eee = and Chere Tp! est, cere to have ha slr ghee Fair th oxo of RA Wemtnre, Wi a2 ata Ios he ncamgs of tomacig te poate To slate Inert In Wingo, we dab a ee fey Ast sare dene stl Li Bypass 2:40 5 capa of Gres A Erg can of ese ‘al ino bs A aot: masieeion re ih oor iy esr emda sek ee ely 1 eeneatonh (comm ed Lu eae a alr hor ir rosea sl aupegiaugi see he ae A ‘asl expansion at presi cls ve a ow sams besoin scone rhs (C1 ely tt Trivedi he tin ates paso Fee hereto 3 Thicpmeee Selly ke pce iho er ed Kes. or WS est melee wid (820, allele 92 ene eged p tad 16 spose ees peers One ‘aks pay ells MBS ier by pom ind EAC to assign atel ew eter orgie in rel wi ror ey rae rscien, The cele nt meet son ofc el In isola ence PRA hase ocr wi I eb pun (PIE). usw bee ee Totty eof een 9 AR sgt, {irowervnes sine Se fin eu Cle Ben se facia) Imseru oe i an pul ane ee me rome or Bo Sisn tect ded moth ere eet iat Kero traterln Biles eDN ae he Seger rhe, ly and DA ee Aroma oh iebachiabontens beet tigany ve oe SeStr iam tests tests be sree tte abit encntoetantensciyes nae emir Wei re grentle shee oes neyo here Beaver poe Ween on carding shorter {Walon apo (ayant We evogsn rpenealomee RE Sli ale ce she anno ts iuacies Pao MRSA HH Se fe eaceke aii eevee once Men ee mrtg che mie Tk 2 ee Ezeh sah erent Preterm oer, CHETEPLIGCA soils Cra bole The meinD'. 2m Melanyeh nceehele Sats eatesraeysaa ISA MELNCAP Co Gemwen x Dawe and Tipe (tne cnc grth Thoda ar gree pg oo ‘vere evoo A casas ef 25 ow Bish roid 5 tls ae elas, even afer diy of amor. Tessing the sctitn of ASA ToS a BC OT ga Ay hoe SG, fps : ] tts Tests ec sat shared unl he 72 Jac tsa tts acc nf eth aio at lad 2b 18 Wis, abe by 92 Bars wae 085 oor eal’ conamet ke eo cue Lhe pooner moh eae te ona 9 MBA wh rope rem hen Ts lag gona ego se a eli of PSA a Al 2 ‘Sore tsi wae heie bs arse oF guhth RE 5 ISA Supproné P84 IVA and Peowin epee in LNCAP Cele The econ FPS! MPA VR snes {Mah rsa igh of by 2A roche oa Pe vA al ers a PSA EDA wa aoe i 28 se agonne Yo MBAsshe MRS lial oppel = SAMI A wat depress seosaatog of SA a De beson 2 se Jp I su oe pena efor apse MISA 1 sions 23 ya MEA re PSK NA el 98 Thee of MSA hed na etm lpr Te mi ‘Seanad baie fore any deck are i pw. Te ‘Game er Psa gt el bv MEA #5 Eo deri be ‘Westen Brean sho in a, 26 poe! Tet pe “uso ls Sn! tone Je FSA pate ee SPH S135 UMA, AcS Je cE Pe vel 9 PRA pra cre Seer lor hls deri, The epermer: ewe a (Ser darsuiveiee iat IPFRS. wal no sano ate eta eminem wc clare = x al FS caning Mma RSE ‘ep seks og The renzo of WA tha oy MB ag Een 9d ad tas, ae casey Hal te towed the FBS tre (Gx nrstoun Se Suprees AR mRNA and Prati Kxpeen ia LNCAP (cae thasapcnie of PSA a lnorate bat fe DEA, WD) [ee ligtsacne rane ren fees Dn et ey ee FREICM. ge shows te fae ous wpe In BSA, ‘Nuh al Latha sw 308 ace in ASIN. The ‘se et nae te nS or ae hae