DTIC ADA423279: Humanizing the Mouse Androgen Receptor to Study Polymorphisms and Mutations in Prostate Cancer

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Andzagen receptor (AR) plays a eriticnl role in prostate oncogenesis. Allelic variants of AR. particulaty in,

| length ot on N-terrsivo} glutamine {Q) ir, are aciated with distinct risks of @iseuse. Mulgan of AR in
4 tumors may err into resistance to tescment and androgen independsuce. Lnniston and progression have been
| ditticuteto study de to lack of early disease samples and lack of animal models. ‘This has been partly overcome
| ith transgenic monsc tumor medels. However, mousc AR differs significantly fiom hurnan in the N-termimas._ |
In oxdor to critically evalvate the role of the polymorphic gtitamins tract in disanse, and to identity elevanc sites}
in he N-terminus whose mutation can lead io andogen-inicpandent AR function, we have “humanized” the
rmousc by converting its androgen recopter gene to the human sequenoe. We have donc this by homologous
recombinstion in embryonic stem ccls,inoducing AR alleles with 12, 2b, orl glutnmines, Mice bearing the
“veil typo” allele with 21 ghatamines arc normal by all ications, including misroarray analysis, Hoover,
expression of specific androgen target genes suggests sue distinctions exist. Thia will be explored in detail by
‘examining lumor progression and by sequencing AR cDNAs from tumors of oarated vs. intact mice.

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vITLE Mumacizing Le Neuse Androgen Revestor to Scudy
Polymorphisms and stations in Prostate Cancer

PRINCIPAL, INVESTIGATOR: Diane M. Robins, Ph.D.

CONTRACTING ORGANTZATTCN: university of Michigan

Ann Arbor, MI 48109-1274

PE GF RRBORT:

EL FOR: U.S. Azmy Medical Resesrch and Materiel Command
Foxt Detrick, Maryla:d 22792-5012

SISTRIEUTION STAYEMENT; approved for Publis Release;
Distribusion Untinited

tae views, opiniens and/or Cindings contained in thie repsrc are
khose of the sucaor(s) and should not be construed as an official
Departmunl of the Army position, policy o: decisicn ualeas eo
Gesignated by othe drcunentazion
Cover

SF 298

Table of Contents

Introduction

Body

Kay Ressarch Accomplishments

Reportable Outcomes

Conclusions

References

Appendices

‘Table of Contents
Annual Report, VAMD17-02-1-0099 January 2K
INTRODUCTION

Both the development and thr progression of proslale cancer (PCa) depend ort
genetic and environmental factors that are poorly dovined. One thing all furmore share,
hrowover, isan intial dependence on androgen for growth [1]. Polymonphisns ir
andeoges eveptor (AR) may impact risk of disease, and somatic mu alioas may affect
Progression and responsi:té eezapy [2-4], fn almost all cases, endncrine therapy is
Inially sueccasful, bot tumors ultimately become endrogertindependen: and eeeist
further treatment, Despite andragen independence, A& levels in the tumor remain high:
and the AR signal:ng pathway is Intact, revealing, a continued role of AR in the cliseas:
pincess. AR molecular genetics may be informative for two crucial problems in PCa: 1)
How do polymorphisms ie AR lead to groatcr risk of disease? 2) Haw do somatic
mutations in AR daring tamor growth eitcumvent hormone ablation? Ehis project
‘direc thane questions (or De human receptor in a transgenic mimnese prostate cance
adel, allowing initiation, progression and treatment of disease te be imfygrated
experimentally. Flucidating the mecharisins by which genctic variants alter AR aclioa
will validate their use as molecular markers in ireatmeni, and, ultimately, may reves’
rovel targets for therapy.

sovy

‘Que hy pothesis i thal genelic variation in human AR affects the initiation and
progecssion of prastate cancer. Coemline vaviation may affet initiation or aggressivity
Sfearly clisease, while somatic mutations may drive andzoger-indepandent growh. To
confirm this hypothesis, our fies inajor objeclive was to replace the mouse ARR with
Inman scquemces to study their efforts im the mouse in general and in a transgenic
tumor madi (Transgenie Adenniearcitursa of the Mouse Prostate, or TRAMP [SI]. At
implicit suh-hypathenis ts that AT species difforenecs impact differential PCa
susceptibility. Since Q (glutamine) tract length eaters into PCa risk, we proposed to
compare varying length Q tract hAR alletes, We alsa plan t compare auatalions arisine,
inandrogen-dependant vs, independent disease, fo ihentiy sites correlating witk AR
fonction. Our aims under'ying the Statement of Work remain ax fallws

Aim. To study the rolein PCs of polymoephisms in human Ar, m4r will be
"humaniced” by homolegiuss eecombination in embryonic stern cells, to create throe
Winas alleles differing in putamine sract lengta (12Q, 210, 41Q). Differences in
androgen action (fertility, belkavior, wiuleculae markers) and spontaneous prostate
cancer will be studied in mice with himar allole

Aim IL To determine the role of human AR variants an PC initiation, Amr alletes
‘will be placed on the TRAMP background for transyene-induced icogenesis. Elfect of
the Q eact will he assessed on peos-ale palhophysialogy and gene expression by CDNA

Aim ILL To dele-mine the role of AR variation on PCa pmyrsitn, spontaneous
‘mutations will be identified in AR DNAs [rom castrated {endragen-indepondent} vs
intact iYetay-TRAMP mice, ‘he effect of mutations will be determined by introduction,
into ARs for transiection analysis, with aad without coactivators. The effoct of
_ulalions on the oncogenic potential of prostate cells wil: be lested by tumor formation
dnd wnctascasin it SCID aie

Each Aim corresponds to a Task within Hu: Statoment of Work, We are
progressing well, but a hit hehind schedule, ducin part to ciff-cullice vith one of th
hace Q tract alleles. ‘Ike 21 and 41 Qh/MAR mouse strains wete created with litle
problem, kut 'S eeSis containing the 12 Q allele had amore cifficult time going
germline. This was probably more a bit af bad luck rathor than a biologival differeace
‘worlh noling, as these cells finally have gone germline {fourth ES clone lested) andl the
sesulling mouse slzain secavs fine. However, it wil stil take many months lo genetate
Uinough mitt: oan the exons tu TRAMP lo generale lumors to compare to the straina
with 21 and 41 Qalleles, ane thos we will fel requesl an additional yea: extension ab
ru additional evst

As described previously for the 22 Qh/mAR mice, which heve naw beer: in
‘existence for nearly two years, hie AF Qhoinnzygous females as well as males showy 10
differences in gross appearance, behavios or ferhlity rom wild type lilte-mates, Mice
of these lixeages are being generated for aging studies and ta track proslale physiology,
over ike next two years. Flormene assays for (estosterone and estreygen ate being,
pesiormed to tesi any differences in feedback regulation. Andmyen tang: genes in
Several tissues are being assessed for the 41 (mice, as they were for the 21 Q ice,
swhoce we formd sorae expressed at highee levels than in wild type mice.

“This mew have alan been cronsud onia the TRAMP background. At the aye of
ten weeks one cohort (svelve mice) was castrated and another Fett inlet, Tumors bom
these mice are bemg, collected, wither when she tumoe becomes greater then ane
wtor by pelpation, ox when dhe ries ve 26-90 weeeks of age. AR DNAs will be

jpaedl From theve fame for mutations ecvarring with progression. Tumors of 4) Q
“ivle ioe ave inthe process of being collected over Hv next few mens, but Lhe
tumors of the 21 Q mice have all been harvested, While molecular anatysis is ongoiny
there sre some interesting results with respect to tumor pragressiim, deseribed below

As oted by ofheas working in the TRAM model, castration (ox treatment with
Antamide) may delay but does not prevent prostate cancer, for aboul 70%: of the treated
mice [6]. However, m about 30% of the mice, a more aggressive disease iniates in the
face of androgen ablalion. This finding bears remarkable simiarty to results of the
SWOG inal in which men were treated with fmasteride [7]. Tumor progcession is Uhus
heterogeneous in ‘TKAMP mice as itis in men, despite the genetic homoggnity of the
mouse model. When TRAMP mice beating the 21 Qh/mAK gene were tested, we
found no remarkable differences in disease progression in intact wild type vs
Inamanized mice. Howeve", in the castrated gronp, the humanized mice showed a
significant inerease in aggressive disease, This is based upim mice requiting cuthannsia
tatlies than the29 woe expertenol endpoint hich was’ out of 1 me forthe
‘wild type group compared to 5 oxt of 13 forthe humanized mice. Molecular analysis of
AR raututions arising, in these mors is eaerently szudesway.

In sum and in accord with our original plan, the second year uf this project has
lnsgely been spent in establishing le third mouse strain for analysig, characterizing the
straing ata grose level, and genercting and collecting tumors from the TRAMP cross.
‘These ines in fac [unstion as predicted, in bviny physiologically normal but haeing
subtle phenotypes when hormonal effects axe examined in greater detail. We are
particularly excited by the apparently great ineweassin aggtessive disease with the
hurnanized allele, as tve would predic: forthe stringer human than mouse AR, Thus
saw are eaciuiraged that these mice will be valuable for assessing the role of ancogen
receptor in prostate cancer initiation and progeession, may provide etter subjects for
ppecelinical esting, and may lead to nev reatments for disease.

KPY RFSRARCTI ACCOMPLISHMENTS,

+ Correctly targeted BS cell lines with 12.0 and 41 Qhumanized AR acces were
successfully incorporated into mouse blastocynts ancl lod lo creation of rama
slrains conlaining human AR sequences in plzcc af mouse, These strirs ar:
gxosely normal and are undexgoing further characterization.

+ Band 41 Qhumanized AK mice were crossed with TRAMP io generate
prostate tumors, The 21 Qtumarshave all been collected, andl in the sooet Cat
twas castrated appear to be a marc aygressive form of disease then in wild type
littesmates, confirming that the humion SR gene has a sublly distinet activity or
funetion than the mouse,

REPORTABLE OUTCOMES

We are currently preparing a manuscript detaiiing the creatinn znd peeliavinary
physioloyivad characterisation of fhe “huraanized” AR mouse, but are waiting foe the
three alleles to be of comparable characterization. We have: >resenied some ofthe data
locally, as well as at the Socond International Conference em Prostate Cancer Research,
ewwa City, 10/12/02, and al the iniorprostate SPORE Conference San Francisco,
12/5/02. ‘his mouse mode! will alsa be used for other experimients proposed as 4
project within the University of Michigan SPOKE in Prostate Cane to determine
cltects of specific antagonists (fulami, bicalutamide) on incidemes of mutations in the
humanized compared to mouse AR.

CONCLUSIONS

‘Minis stranns carrying, human rather than mouse AR sequences have been
cvnsteucted, This will allow direct testing af the ole of AI glutamine tract variation in
initiation of prostate cancer, which may help clarity contradclory results from
epidemiological studies. Fvther, tomers initiated by tranwgeuss in these mice wll
allow tracking ihe role of AR, and AR mutants, in resistance to antiondeogen therapy
‘and androgen independent growth, Lhe site of mutations im human AR sequence may
lead to downstream interacting proteins that will be novel and more effective tazgets in
new trea'mon| stealepics

REFERENCES.

Tk] Henderson, BE, Ross, R.K. and Pike, M.C. (1991) Teward the primary
prevention of cancer. Solence 254, TI31-1158,
J Tevine, RA, Yu MC, Ross, RK, and Coetce, G.A, (1995) ‘The CAG and GCC
_miceosatellites oF lhe androgen receptor gene are in linkage disequilitivm ir
mon with prostate cancer. Cancer Research 85, 1937-1940

DB] Paimberg, ©. Koiviste, P, Kakcola, L, Tammela, T1., Kallioniem, ©. and
‘Visakorpi, T, 2060) Arulriygen eeonplin gene amplification at primacy
pprogeewsion predicts respunse tu combined androgen blockade as second ine
therapy Zor aetvanced prostate carer. J. Ural. 168, 1982-1995,

I4l ‘Taplin, M4, Bubley, GI, Ko, ¥.J, Small, BJ, Upton, M, Rajeshkumaa, B, and
Rall, 82°. (1998 Seinetion for andtogen zeceptor mutation’ in prostate cancers
teated with androgen antagonist. Cancer Res, 59, 2511-2515,

Bl Greenberg NM, Debayo, F, Finegold, MJ, Medina, D, Tiley, WD, Aspinall,
Os Cunha, GR, Donjacour, A. Ay Matos. and Rosen, (1995) Prectats
cancerina transgenic mouse. Proc, Natl. Acad. Se. US.A. 93, 429-3443,

J6l Gingrich, L28, Barvios, RJ, Kotlan, MW. Nalur, ITS. Cinegald, MT, and
Greenberg N.M, (1997} Androgen-mdependent srosiale cancer progressitin in
the TRAMP model. Cancer Research 57, 1487-4991

17] ‘Thompson, IM, Goodman, PJ, Tanger, CM. eal. (2008) The influence of
finasteride on the devetopment of prostate cancer. NEJM: 349, 215-224.

APPENDICES - none.