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an. Award Muuber: ” DAYDI7-93~1-5430 VITGE: Roles of Stersid Receptor Coactivators in Breast Cencer AL INVESTIGATOR: Moed-Hue ¥. deng, F2.5. CONTRACTING ORGANTZATION: Indiana University Indianapolis, T:dlana 45202-5167 DATE: Jenuazy 2304 PS OF REPORT: SEARED FOR: U.S. Kyrty Medical Research end vateriel Conmand Fort DoLrck, Maryland 21702-50"2 DISTRIEJTION STATEMENT: Approved for Public Re“csse; Distripation unlimited The views, opinions ard/ur {indings contained in this report are those of the author(s) ané should noc be construed as en official Depaztwent of tke amy position, policy or decision unless 50 designated by other documeataticn. BEST AVAILABLE GOPY / 20040922 020 REPORT DOGUMENTATION PAGE cla Sreoise Sou croatia ger AN PENG oe ae es Patras ditr Eran RRR TERR ATES TS ona Sopsary 2068 Boner ap 1900 = Tbe 2003) TT A ETE 5 fotos of stecvia seceptor coactivators in arwast Cancer ewltasoc aie | Meel-Hee ¥. Jose. PEA) 7 FERRE CRATER TO HABE AND RODS "PEREGRINE CHGAMTZATION dndlaca Uatveredey ‘eronr aunioan Teclacmpolian. Totivun 66202-5167 eit ahenedivpyl ede GRORTGRA TAATORIS TE SPOSORIE ASAT [AGENCY AAIIES) AND ADDRESS) ‘AGENCY REPORT HUBER fuse. smy todica: Research and watertel comand Port Detsisk, Maryland 42702-5022 agrvovad for Publis Relesse; Distribetion Unlimited TE ARNT TR BT WORRY Tas pempene of thie atsdy ig to determina the roles of gtareid receptor soactivators in brecat cancer. The acove of the research ‘2 lo avsmvy bie expression and functiona of BRC- Y ramiiy meabare (SkC-1, Gael, 9nd Ais1} in comma: révmazy giana and ia breast tumors, We Foged ther tho oxproszion of 80-1 family namiere vas regulated by ovarian herwones dicing mammary q-and development, We glso foul Unt ATb1 protair wae overexsrensed (n tuntn breast eaicer specinons, as corparet to adjacent nortsl breae= tigevo, In addition, notions of these coactivators save been aUsdies duwing the funting verlos. Ne have constructed adecovisus expensing tron thraa conctiveors, In aésitean, ineredod eigeessisn oF ATBI if, Domes Broust cancer celle eimulated the § phase ectry during cell cyele profession, isdicaticg the involvenent of A2Bl in cell eyele content und cn] Gravis of Nora” iwewst ewrenr calle, Surthemore, ovezexpressch St ALBL dromatical-y inereased the stospuorylation of Akt and AkL Ilasce adbivite in human breast cacear cells BSI was alsa able Lo viiminte c-Src signaling. These otudiee previde imporzant. ineic! Row ATH wocks in aumae breast cancer ce!-s to promote breast cancer procreesia: A SORIECT TER TE ONE OF PRES rene: canses, endocrinology, acenovinus, ABI, §R4-1, caactivatora 2 EAR COO TE REGRET LARSECATION | 18 ECORTTY CASSTICATION | 19. SECURITY CLASSRCATON —| 35 YATPON OF ABSTRACT trolassigied tnelaseitiea Unelagel fied Untinized TN TSE "Sangara Fas 2 ee 207 Table of Contents Cover... SF 298. ‘Table of Contonts.. Introduction... Body... Koy Resoarch Accomplishments. Reportable Outcomes.. Conclusions. At References. 7 Appendlees. Introduction ‘The subjeut of the current studies isto determine che expression and fonctions of steroid ree=pior coactivator] (SRC-1) family masmers (SRC-), GRIP, and ATB1) in norma] mammary gland and in breast cancer. We have evaluated the expression all SRC-I family members iu human breast tumors and fannd that ATE wos overexpressed ia many breast tumor specimtens, ‘The scope of the resoarch is asnursing the fonctions of SRC family members ducing breast cancer progrssvicn. We also assess the Functions of SRC-1 family members by construing senovivus exprensiag ‘coactivators aud detemuine ths moleculer actions of these couclivaloes i uoian breast cancer alls. Body Task E-Cennpleted, Collect n of mammary gland samples bas been completed ‘Task 2: Completed, Weave aalyzeé the exprossion wf SRC-1, GRIPL and AIB1 in rat mammary sland using antibodies specifically geins! Urese proteins. We Gund that SRC-1, GRIPL, ard AIBI levels are slovated in miammory gland during pregoaney. a8 eompared to virgin mamunary (land. Tlis interesting to aote hae SRC-1 was colocalized with csrogen receplor (FR) alpha in rmiunmary epithelium ir pregnant feniale rats and thatthe expression of SRC-I wus regulate by avatian steroid hormones. Interestingly, SRC-1 remsined segeeguled Irom PR olpha in iavoluting mamznary cpitheliwn, GRIPI and ATB) were asm expressed in ral matory epithatial cells, Our alu ayges that regulate athe eapression af SRC-1 may be important fn detsemining the transactivation funedan af UR alpha during the developmcnt of normal usammary cpithelium, “Task: Eliminated per contract agrosment, Task 4: Completed. We have toate! animals with hormanes, and mammary gland samples have been collected Task S: Completed. We have examined the expression of coactivators in mammary gland ypun hormone uestment. Since the expzessiax. of SRC-1 family micmbers was elevated fn pregnant _muummary gland, we speculated fn: pregramey hermes estragen ara progenuertine were able (9 regulate the expression af SRC-1 Iorily meribers. Rats vere ovariectomized 10 reduce the cireu.ating estagen and progesterone levels, Ous wook later. estrogen and prageslerons, slam or in combination, were adinixistrated, Rat mammaury glands were dissectad (or the analysis of SRC= 1 fomily members using western blol snalyyix. ‘We lbund that adncnistraion of estrogen or pmigesterone alone decreased the expression of SRC-1. However, the expression of SRC-1 was tlevuled hy the treatment oCestrager. and progesterone. jection of Ad-CMV-beta galactosidase reporter into macamary glaud for ho assosscont of ER, ransuctivation unetion alsa demonstrated (that treanvent wth hoth estrogen und progesterone wus able to atiruate the ER transactivation anction in macsmnaty gland. Our data suggest that irercased expression of SRC-1 dining rcgnancy was able to crhtanes the ER transactivation fimetion, canuscript is in preparatinn to document these observations. ‘Tusk 6: Completed. We have completed the collect specimens. nd embodkling of umman bresst lamor ‘Task 7: Completed. The immunohisochemical staining of SRC-1 end ATBI in human breast lamors has bgen campletee. AUT was overexpresscl in human breast tunvots, after comparing 122 clinical specimens (tables 1 and 2). We ave facing background problems using 11K2/GRIPL anthody for iemnobistochemical stining. We are still trying to improve this background problem, ‘Task 8: Completed, A manuscript isin prepatstion to dacmment the stininy dats an corrstation ‘vit clinioal pathologteal parameters “Task: Comple:ed, We havo daue immunsiaining of husazn breast cancer MCF-7 vells wing, ‘axi-SRC-. and antiAIBI antibodies. Nuclear staining of SRC+4 and AIB) were vhserved. Twa less deers tre eyluphasie staining was obsorved, This is in contast lo Cre tumoe stsining we have perfornied, Ir several eaxes, we observed the cytoplasmic slaining of SRC-1 and AlB1 io ‘vamor ells in haman breast tumors, We are nat certain whether the hormonal level in patents or ‘ells will influence the localization of SRC-1 or AIBI in vet Yak 10; Completed, To examine the exaeession of p160 coactivators in human hreast cancer cell Fines, we have dine Western bloc analysis for SRC-i, GRIPL, 9nd AID in several hnan breast cancer cell Fines, oth ERe-pesitive (MCE-7, T#7D, ard 7R-75-1) saul LRenezative (HCC1S57, MCTIAA, MDA-Mi.231, MDA-MB-4358, BT-20, and SKI) cells express all throc ccoactivecais, MCF-7 ces expressed 2 much higher level of AIB] among all she eu fies tested, Task 11: Completed, We huve successfully constructed the sense constuet af the udenaviras cexptessing ATR, GRIPI/TIF2 and SRC-1, ‘These viruses arc tegged wich GTP in onder to monitor the infection and expresston cffisieney pT this virus. The canlrot virus AdGH? has also been constructed ‘Tusk 12: Campleted. We have completed the testing for adenoviruses vantructed in tissne culms tells, We have performed Westem blot analysis and arc able wo show that AMGEP-ALB express funcional ALBI, Preliminary dats indicate hat overexpression of AIBI increased the § phsse of cell cyclen T47D cals amel MCP-7 cells (Figure I). Adkltion uf PISK/Aldt inhibitor LY294002 abolished the AIB] enhanced § phase ents (gure 2). Infbction of human breast cancer ees with AIBI increased Akt phosphorylutine (Jigure 3) and Akt kinase activity (figure 4), suggesting the involvement of Akt signaling in mediating AIR copulate call eycle pragressent. Fuatbesmiore, ‘AIB1 fas able to interact with o-Ste in immmnoprecipitalion experiment (figure 5), an important ‘éaptor protein fer growth {nolor signaling, { transtection experiment, AL] aud circ was able to synorgistically activate FR iransuetivalion fonction (igure 6}. ‘These date suggest that cross talk of ATEI signaling, with geovel factor signal’ng is playiug au important role in medinsing AID actions in harmon breast eunce= cells ‘Task 15: Completed. Large-scale prepariaion af udenaviruses :3s been comateted through Cs Teanding ‘ask 14; Completed, We have infused the purified alenovieases wo rat manurary gland for ‘orsig the offeels of ATR «m modulating Et sipnaling pathway in situ. Task 15: Completed, We di Liquid X-gal ssininy irs mammary gland infused with Ad-ERE-bota galactosidase reporier adenovirus tad ndenovinis expressing coactivators. However, we did not 5 see much X-gel shining. this rime, We doa’t xaos why, and we are ou the process ol optimizing, the experimental protocols, Key Kesearch Accomplishments We have demumstrated the eegulation of SRC-1 family members during the developmen of rat smaramary gland = We have demonstrated that AIBY protein was altered in many breast cancer tamors 1 and GRIP. = We have successfully constructed adenovirus expressing AIBI, SR = We have generated mbbit antibody against AIRL = We have demonstrated thar ATR] over-expression could increase the S phase of che eel eycle Adtition of Y294002 was able to abolish the AIBL stimulated ineseate cf S phase entry. We found that AIB1 was able ro ntemsel wilh o-Sre adaptor protein, Transfection of ATB with &- Se enfanced the FR tranentivation Function synergistically in human breast canoer cells Infection of urna hreast cancer cells wit adenovirus expressing ATR increased the phosphorylation of Aktaud Ar kinase activity, 17204002 was able te abolish @e ALBL stinlated increase ofS phase enty, suggesting the irvolvement of prowth factor signaling pathways in mediating ATBI action in human breast cancer cells. Reportable Outeomes Absraets J, Shim, W-S., Tarser, M.A., Samu, Kl, Jeng, MoH. Up-regulation of SRC-I Peoteln and Anciesse of ER in Sita Trewssetivalion Function by pregnancy Mormoues. 83" Annual ‘Mecting of the Endocrine Suciey, Fare, 2001. Oral Presentation. 2. Zhang, Qell, Chang, LY, Viet, B, Stalleup, MR., Fdswurdy, DP., Cheng, L, Goulet, BJ, and Jeng, M.-H. Over-sxprossign of Reveral Nuclear Receptor Cosctivator fro in Human Breest Cercinoma, 82°" Anmual Meeting of the Ensloceine Society, June, 2001 3. Long, X.,Cai, A Vict, E,, Quilliam, L.A.. ani Jeng, M-TL Tnterantion of e-Sre with Stevoid Receptor Conetivaters. Keysline Sympeniuar D4: Nucteat Receptor Superfamily 2002, Late bronk-through aval presente 4. dong, Mol, Zhang, QoL, Long, X. Functions of Esrogen Receptor Co Mecting, 2002, « GouletsR., Slee, G, 14,1. and Quilian L ulatrs i Breast Cancer, DOD Era of Hope Zharg, 1, Chang, L.=Y., Long. %, Cai, A. Vie, F, Kau, C., aul deag, MoH. increased Fapression nf ATT] Stimululed Akt Phosphocylation and Cell Cycle Progression in Homan Treast Cancer Cells, 85% Amal Mectieg ef the Endocrine Shsiety, June, 2003 6 ral presentation Cone ‘Dating this funding period, we have completed the lisks snd evaluated the expression and imolecutar actions of SRC-1 farily merners. We have demonstrated that the eapression of SRC-1 ‘amily members was regulated during the developinent of tke matnmary gland. In addition, ALB van ovetexpressed in human broast more. More finkintantty, AIB1 can activate e-Sre and Akt singling patbyrays, The intoractions of AIBI with ¢-Sre and Ake signalings alsa provide important insights of the molecplerretions af AIBL in human breast canesteclls, The generation cf adenoviruses expressing conetivatens will provide us a valunblc tool fo astess the fusetional ale cof SI{C-1 feily members in FR irananctivation function in mammary gland and io regelating breast eanoor cell proliferation, Our curent studies suggest that overexpression of ATG] protein ray conte ty inensaved § phase and subsequently increased cal] praliferation in buman breast spithelisl coll, References 1. Shim, W,-S, DiRenaw, 1, DeCapeo, J. A, Santen, R, J, Brown, M1, Jeng, ML Sogregrtion ol era receptor casctivato-] from steroid eveplors in mamraary pitheliam, Processing of ths Nal'onal Acudemy of Sciences 96{1) 208-213, 2909. 2, Jong, MHL, Kao, C., Sivaranan, L., Kmacik, $., Ching, 1. W. K., Medina, D., Comnecly, 0.M,, and O'Malley, B. W. Roconstition ofesirayen deperitet transcriptional activation fan adenovizal target gone in select regions of the rat mammary gland. Enegerinelogy. 139(6): 2916-2025. 1998, 3. Shim, WS, umer, M.A., Santon, RF, Feng, MII. Up-tegutation of SRC Protein and Inerease of ER in Situ Transaetivetian Tunetion by pregnancy Hormones. 83° Annual Meeting of the Endocrine Society, June, 2001. Oral Presentadon. ang, Q-H,, Chang, L-V.. Viet F. Stalleup, MR, Rewards, D1, Chong, L.. Goulet, RU. abd Jeng, M.-H. Over-espression of Several Nuciear Receptor Coactivator Proteins in Human Breast Carcinoma, BY Anaual Mecting of the Kndscrine Sociely, June, 2901 5. Loog, X., Cai A. Vieth, B.. Quill. T.A.,und Feng, M-IL Lateraction of c-Sre wit Steroid Receptor Conctivaters. Keystone Symposium D4: Nuclear Receptor Super iil 2000. Lave break-thzough eral presentation Jeng, MH, Zhang. Q-H., Long. X. Goulet, R., Sledge, G. Li, Land Quilliam L. anetions of Estiogen Receptor Coregulaines im Breast Canecr, DOD Era of Hope Mcsing, 2002 7. Zhang, J, Chmg.L.-¥., Tamu, X., Csi, A. Wieth, Kao, C., and Jong, MH. Ineneasec Expression of ATBI Stimulated Ale Phosphorylacion and Cell Cyclo Progression in ura Breast Cancer Cells, 85" Ana! Mer:ing ofthe Frulvecine Society, June, 2008, ‘eal preseuration, Appendices Uo tables, 6 figures and four abstects Table 1 Clinicopathotogical factors of the breast carcinoma patients Factor Number Percentage (%) Age <50 39 3197 >=80 3 68.8 Tomor size <2om or 53.27 2.1-Sem 4 41.12 > Sem 6 561 Lymph node Negative % 5385 Positive 48 46.15 Staging 1 35 35.00 2 # 400 3 u 41.00 Grading Well oN 1068 Moderate 48 4151 Poor B 41.75 ER status Negative 36 2951 Positive 86 7049 BR status Negative n 58.20 Positive 51 410 Table.2 ABI Immunoueti ity of Normat Breast tissue and Breast Carcinoma AIR] Staining latensity a “o L 2 Incas reduction iesne 9 1 6 2 ° adjacent Breast sue 105 37 st 7 1 gainoma 122 18 3 3A a