TITLE: Mechanisms of Radiosensitization by the Neurotensin Receptor Antagonist SR48692 in Prostate Cancer Models

Transcription

1 AD Award Number: W81XWH TITLE: Mechanisms f Radisensitizatin by the Neurtensin Receptr Antagnist SR48692 in Prstate Cancer Mdels PRINCIPAL INVESTIGATOR: Jarslaw Dziegielewski, Ph.D. CONTRACTING ORGANIZATION: University f Virginia Charlttesville, VA REPORT DATE: April 2009 TYPE OF REPORT: Annual PREPARED FOR: U.S. Army Medical Research and Materiel Cmmand Frt Detrick, Maryland DISTRIBUTION STATEMENT: Apprved fr Public Release; Distributin Unlimited The views, pinins and/r findings cntained in this reprt are thse f the authr(s) and shuld nt be cnstrued as an fficial Department f the Army psitin, plicy r decisin unless s designated by ther dcumentatin.

2 REPORT DOCUMENTATION PAGE Frm Apprved OMB N Public reprting burden fr this cllectin f infrmatin is estimated t average 1 hur per respnse, including the time fr reviewing instructins, searching existing data surces, gathering and maintaining the data needed, and cmpleting and reviewing this cllectin f infrmatin. Send cmments regarding this burden estimate r any ther aspect f this cllectin f infrmatin, including suggestins fr reducing this burden t Department f Defense, Washingtn Headquarters Services, Directrate fr Infrmatin Operatins and Reprts ( ), 1215 Jeffersn Davis Highway, Suite 1204, Arlingtn, VA Respndents shuld be aware that ntwithstanding any ther prvisin f law, n persn shall be subject t any penalty fr failing t cmply with a cllectin f infrmatin if it des nt display a currently valid OMB cntrl number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE (DD-MM-YYYY) REPORT TYPE Annual 3. DATES COVERED (Frm - T) 1 APR MAR TITLE AND SUBTITLE 5a. CONTRACT NUMBER Mechanisms f Radisensitizatin by the Neurtensin Receptr Antagnist SR48692 in Prstate Cancer Mdels 5b. GRANT NUMBER W81XWH c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Jarslaw Dziegielewski, Ph.D. ; JD5KF@VIRGINIA.EDU 5d. PROJECT NUMBER 5e. TASK NUMBER * 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBER University f Virginia Charlttesville, VA SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR S ACRONYM(S) U.S. Army Medical Research and Materiel Cmmand Frt Detrick, Maryland SPONSOR/MONITOR S REPORT NUMBER(S) 12. DISTRIBUTION / AVAILABILITY STATEMENT Apprved fr Public Release; Distributin Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Radiatin is ne f the mst used treatments fr prstate cancer. Hwever, it causes several negative sideeffects, including the damage t surrunding nrmal tissues. An agent which selectively sensitizes prstate tumr but nt nrmal prstate culd imprve the therapeutic rati f radiatin. Neurtensin is a ne f the factrs secreted by neurendcrine cells in prstate, which stimulates and prmtes cancer cell grwth and prliferatin. In this prject we are investigating if the inhibitin f neurtensin receptr by SR48692 drug culd sensitize cancer cells t radiatin. SR48692 activity was measured in PC3, C42 and LNCaP prstate cancer cells, as well as in RWPE1 nrmal prstate epithelial cells, using clngenic survival and grwth inhibitin assays. PC3Mluc rhttpic xengrafts in nude mice were treated with SR48962, radiatin, r in cmbinatin, and tumr grwth was determined by biluminescence imaging. Our results shw that inhibitin f neurtensin receptr by SR48692 effectively sensitizes human prstate cancer cells t radiatin, while the effects n nrmal prstate epithelial cells are minimal. Imprtantly, the cmbinatin therapy (SR48692 and radiatin) suppressed xengraft tumr grwth in nude mice significantly mre that either treatment alne. Our study suggest that NTR1 receptr, r neurtensin signaling pathway, are viable targets fr cmbined chem/raditherapy f prstate cancer. 15. SUBJECT TERMS raditherapy, prstate cancer, drug develpment, neurendcrine signaling 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT a. REPORT U b. ABSTRACT U 18. NUMBER OF PAGES c. THIS PAGE U UU 10 19a. NAME OF RESPONSIBLE PERSON USAMRMC 19b. TELEPHONE NUMBER (include area cde) Standard Frm 298 (Rev. 8-98) Prescribed by ANSI Std. Z39.18

3 Table f Cntents Intrductin 4 Bdy 4 Key Research Accmplishments 10 Reprtable Outcmes 10 Cnclusin 10 3

4 INTRODUCTION Prstate cancer is the mst cmmn cancer in men in the United States, and th secnd leading cause f cancer deaths. Raditherapy f prstate cancer is limited by b I th radiresistance f prstate cancer cells and the adverse radiatin effects n surrunding nrmal tissue. Thus, an agent that radisensitizes prstate tumr cells but sparefs nrmal prstate tissue wuld have clinical significance. Based n ur previus studies qncerning the rle f neurendcrine cells (NE) in prstate cancer, we hypthesized that SR48692, a specific inhibitr f neurtensin receptr 1 (NTR1), (1) will radisensitize cancerl cells and tumrs, (2) that nrmal prstatic tissue will be spared in-viv, and (3) that the mechanism invlves EGFR Tyr845/Src/Stat5b. These hyptheses are tested in tw SpecificlAims: (1) t study the radisensitizing prperties f SR48692 using andrgen-sensitive and fndrgeninsensitive prstate cancer mdels vs. nrmal tissue effects in-vitr and in-viv; and (2) t examine the mlecular mechanisms f radisensitizatin by SR48692 in human prstate cancer cell lines by studying nvel effectrs dwnstream f the neurtensin rec,ptr (NTR1). Successful cmpleting f this prject will allw us t develp an efficient radis nsitizing agent, with ptential clinical applicatin in prstate cancer raditherapy. BODY This is the first year reprt fr grant W81 XWH cvering the initial phase f the prject. During this phase we have cncentrated n Specific Aim 1, investigating the radisensitizing activity f SR48692 in bth in-viv and in-vitr mdel systems. I Fr in-viv studies nude mice harbring human rthtpic (intra-prstate) xendrafts were used. The xengrafts were established frm PC-3M-luc cells, a metastatic variaht f human prstate adencarcinma PC-3 cells, engineered t stably express luciferase. Tis system allws fr nn-invasive cntinuus measurements f cancer vlume/size, even Ifr xengrafts lcated inside the bdy. In additin, ur preliminary experiments denstrated that in this system biluminescence intensity crrelates very well with tumr sizl' as measured by calipers. NCI nu/nu mice aged 5 weeks were injected in the psterir ventral lbe f the rstate (rthtpically) with 5x10 6 PC-3M-luc cells. The mice were divided int 4 grup (cntrl, radiatin nly, drug nly, and IR + drug). On days 4-8 pst-injectin, the grupsjreceiving SR48692 were gavaged with 25mg/kg drug. Mice receiving radiatin were subjected t a 2.5 Gy dse f inizing radiatin n days 5 and 7 pst-injectin. The mice were werf anesthetized, injected with luciferin and imaged n a Xengen IVIS system every 7 days pst-injectin. As demnstrated in Fig 1 mice receiving the cmbinatin therapy (SR48692 an I radiatin) shwed significant reductin in tumr size ver either treatments alne. Interestingly, the radiatin alne regime used here delayed tumr grwth fr apprximately tw 1eeks, but did nt prduce significant decrease in tumrs number r size. Hwever, the cmbiratin treatment with SR48692 nt nly delayed the first ccurrence f measurable tu rs, it als seemed t eradicate tumrs in sme mice. Further studies t cnfirm these bs rvatins are under way. 4

5 A cntrl7d cntrl42d SR486927d IR 7d SR d r. i w IR 42d,,'3',0:;.J IR + SR 7d IR + SR 42d B ----,_.._ PC-3M-luc ORTHOTOPIC 6x10 9,-- " ".,-,.""""".-."".", ",,.-, "----' -.-.",,. - 5XI0 9,0" cntrl, SR IR IR + SR48692,/ x ::> «= c.s 2 X c Q , 40 Days pst irradiatin Figure 1. SR48692 sensitizes human rthtpic xengrafts in mice in-viv t therapeutic d s f inizing radiatin. Tumr size was measured by nn-invasive biluminescence imaging 5

6 In in-vitr studies in this first phase we have cmpared the radisensitizing prplerties f SR48692 in prstate cancer cell line (PC-3M, highly metastatic human prstate adencarcinma cells) and apparently nrmal human prstate epithelial cells (1WPE-1). Bth cell lines were treated with SR48692 at 1 IJM, the drug cncentratin kn t efficiently blck neurtensin-dependent cells' grwth stimulatin, as established in ur pre iminary experiments. Fllwing 24 h treatment the cells were irradiated with x-rays dss in the range frm a t 6 Gy and re-plated fr clny frmatin. Clnies cntaining mre tha 50 cells J were fixed, stained and scred, and the surviving fractin was calculated accr ing t standard methd. As shwn in Fig 2A SR48692 effectively sensitizes prstate cancer cells PC-3 I t inizing radiatin at all studied dses. Based n the shape f c1ngenic survival curve IDbtained frm drug-treated cells, i.e. steep curve lacking brad shulder in the lw dse rang, we expect that SR48692 activity culd be mediated by ne f the fllwing mechanisms, III f which result in steep shape f c1ngenic survival curve: (1) inhibitin f DNA damag repair, (2) synchrnizatin f cells in M phase f cell cycle, (3) efficient inductin f apptqtic cell death, and/r (4) any cmbinatin f the abve. These pssible mechanisms f SR48692-induced radisensitizatin are currently under investigatin. I Imprtantly, pre-treatment with SR48692 des nt have any significant effect 0 radisensitivity f nrmal epithelial cell line RWPE-1, as shwn in Fig 28. This striking difference between nrmal (RWPE-1) and cancer (PC-3M) prstate cells is emhasized n Fig 2C, by cmparing survival curves f bth drug-treated and irradiated celllins. As demnstrated n the graph, at the clinically relevant dse f 2 Gy, the difference between surviving fractins (SF2) in RWPE-1 and PC-3M cells treated with SR48692 an irradiated is apprximately 2.1. Therapeutic gain f this magnitude has high clinical significa l ce fr raditherapy f prstate cancer. The difference between these tw cell lines can be explained by ur hypthesis that nly cells expressing neurtensin receptr 1 (NTR1) will respnd t SR48692 treatrllent. Our preliminary results clearly demnstrated that nrmal prstate cells (RWPE-1) d<b nt express NTR1, as ppsite t cancer cells (PC-3M). Since there is evidence that majri'y f human prstate tumrs express NTR1, while nrmal surrunding tissues d nt, SR48692 culd becme a very imprtant clinically radisensitizer fr prstate raditherapy. I In additinal experiments we have tested the pssibility that SR48692 alne isyttxic t prstate cancer cells (nt the slight delay f xengrafts develpment in mice tr ated with the drug alne, Fig 1). PC-3M cells were treated with the drug at dses ranging fr 1m a t 10 IJM fr 24h, and then cunted directly fr shrt-term grwth inhibitin assessment. IF,'r lng-term c1ngenic survival the cells were plated at 100 cells per dish, treated cntinul!lsly fr 7-10 days, and the surviving clnies were stained and scred. As shwn in Fig 3, tie drug alne is relatively nn-txic t PC-3M cancer cells. 6

7 A PC-3M ". " '" 0,1 ctrl +SR48692 II-lM '., B t '...,-----, Xrays [Gy] RWPE-l ,:... " '.> ctrl +SR48692 II-lM c ,-----r'-----"-----,, X rays [Gy] l ,-----,----"'---, Figure 2. SR48692 sensitizes human prstate cancer cells (A, PC-3M) t inizing radiatin in- 'fr, but des nt have any significant radisensitizing effect in nrmal prstate epithelial cells (8, RWPE-1), ( ) Cmparisn f radisensitivity f SR48692-treated nrmal (RWPE-1) and cancer (PC-3M) prstate cells, 7

8 1 PC-3M cells c 0.75 :p u u.. C'I c : c: :::l Ul _..- Clngenic Survival (cnt) --0._- Grwth Inhibitin (24h) O-j------, , r SR48692 [11M] Figure 3. SR4869 alne is relatively nn txic t PC-3M cancer cells. We have als started t explre the rle f cells/cancers' andrgen-dependenc in their sensitivity t SR48692 and radiatin treatment. Three cell lines were selected fqr the preliminary experiments: (1) andrgen-sensitive LNCaP, (2) andrgen-independent C4-2, and (3) nrmal prstate cells RWPE-1. Cells were grwn in medium depleted 0 andrgen (and ther sterid hrmnes) by charcal treatment, and supplemented with R1881 (andrgen substitute), NT (neurtensin), r full fetal bvine serum (full medium) Fllwing 24 h incubatin the cells were treated with increasing cncentratins f SR48692, nd the cells' viability was measured 5 days later by XTT assay. As demnstrated in Fig 4 n mal prstate cells (RWPE-1, Fig 4A) as well as andrgen-independent cancer cells (C4-2, Fig 4C) grw relatively well in andrgen-free medium, and d nt respnd significantly t SR In cntrast, LNCaP cells (andrgen-sensitive, Fig 48) nt nly require andrgen ted prliferate, but als their respnse t SR48692 treatment is mre prnunced. Interestingl, stimulatin with NT can induce prliferatin (up t -50% f nrmal rate) in andrgen-deplerrd LNCaP cells. The fllw-up studies testing the radisensitivity f LNCaP, C4-2, PC-3M nd RWPE-1 cells in the presence r absence f andrgen are in prgress. 8

9 A /' RWPE-1 /... -'"...-.._-... '"'...,... / i"/ E 6' 6 0 u X X X : X '.,2.,Ii e C- 0.5 U B 2.0 X +l%css 0 + R1BBllnM 6 + NT loonm + 10% FBS 0 0:1 i SR48692 [MJ [NCaP -.:: u '._.,j _ _- c '" 1.0 w '"e C- C O u x, 0.5 X +1%C R1BBl lnm 6 + NT loonm -a- + looffbs SR48692 [MJ C4-2..._...-._ _,._._..._-._-..._.._..- X ----'..., w e'" 0.5 C- 0 t. O U X 0 X %C55 + R1BBllnM 6 + NT loonm -.- +lo%fbs 0.1 SR48692 [MJ Figure 4. SR4869 cyttxic activity n prstate cells differs depending n their andrgen depe dence. 9

10 KEY RESEARCH ACCOMPLISHMENTS SR48692 sensitizes human prstate cancer xengrafts t inizing radi tin in viv SR48692 sensitizes human prstate cancer cells t inizing radiatin if vitr SR48692 des nt sensitize human prstate nrmal epithelial cells td inizing radiatin in vitr (the therapeutic gain f abut 2) REPORTABLE OUTCOMES The results btained during the first year f this prject were presented at the 100 th Annual Meeting f the American Assciatin fr Cancer Research (April 18-22, Denver] CO) [1]. In additin tw presentatins describing ur results were accepted fr presentati during this year annual meetings f Radiatin Research Sciety (Octber 4-7, Savannah, <BA) [2], and American Sciety fr Therapeutic Radilgy and Onclgy (Nvember 1-5, chipag, IL) [3]. [1] G Amrin, B Pembertn, M Dunlap-Brwn, S Parsns; Neurtensin recept6r antagnist SR48692: ptential radisensitizer in mice. [2] J Dziegielewski, BL Pembertn, ME Dunlap-Brwn, JM Lamer, SJ Parsns, GP Amrin; SR48692, a specific neurtensin receptr (NTR1) antagnist, sensitizes prstat cancer cells t inizing radiatin, in bth in-vitr and in-viv mdels. ji [3] J Dziegielewski, BL Pembertn, ME Dunlap-Brwn, SJ Parsns, GP Amrin, JM Lamer; The specific neurtensin receptr (NTR1) antagnist, SR48692, sensitizes pr 'tate cancer cells t inizing radiatin, in bth in vitr and in viv mdels. CONCLUSION The results btained during the first year f this prject cnvincingly demnstrae that SR48692, a specific inhibitr f NTR1 receptr, sensitizes human prstate tum rs and cancer cells t inizing radiatin at dses used in clinical raditherapy. At the same tim SR48692 des nt sensitizes nrmal prstate cells t radiatin. The experiments aiming t elucidate the mlecular mechanism(s) f SR48692 radisensitizing activity and selectivit are in prgress, and will prvide a slid fundatin fr future selective raditherapy f human prstate cancers. 10