DTIC ADA430062: Regulation of TRAIL-Mediated Apoptosis in Prostate Cancer by Overexpression in XIAP

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Regulatfon of TRATL-Mediated Apoptosis in Prostate Cancer
by Ovexoxpreeaion of XTAP

PRINCTPAT TNYRET-GATOR: Renjanin Bouayida, Ph.D.

CONTRACTING ORGRN-ZATION: Universizy of California, Los Rngeles
oa Angeles, California sc024-:40c

eMEGHT DA, Jaruary 2004

REPORT: Rnnual

FREPARED FOR: U.S. Army"Medicel Xesearch and Materie. Connand
wt Becrick, Maryland 2:702-5912

LISTRIDUIION STATIMENT: Approved for Public Release:
Dianribusior linlimited

‘whe views, opinions and/or findings contained in this repor: arc
those of the author is) and esould not be construed es an official
Separtment. o* the Arny Position, policy or decision mess so
designated by other documentation.

20050218 085
For Approved

| 2 __ REPORT DOCUMENTATION PAGE OND No, 674.9988

rainy Uae ONLY ‘Enaport bate REPORT TYPE BRD BATES CONERD
‘own ben Sanusry 2004 Bezual (25 boo 92-14 Dee 02)

Sugulation of TRL Nediated apoptonin in Proe
by Oveceupreseion of XIAP

cancer | Samoa" 92 20922

Sungawin Bonavida, mh.D.

[7 RERFORNG OREARTARTION NAME) AND ADORESSTEST 1 PORFGRAG OREANTEATION
Usiversity of californta, Los aogeleo RePoRT MUMBER
Uw Bngeies, CeliLornea’ s0nz2-1606

8: bbonayidaerodrot ucla-odt

DOENCY NAMIES| AND ADRESSES) [RRENEY REPORT NUMER
0.8, acy Yadlaal esearch and materie= Command
Port Detrick, Morylana | 2=702-5212

1 BOPP RENT NOTES

[ i STRRTHON TAVATLADLTY STaTERONT YEE OTTO SOE
Approvee tox Gablie Release; Distribution UnLimized

RETR Ra WOT
‘Te feilureto erosions dvenced prose cancer thats sesitant to conventional hotnpss bax Leto the exptortion,
of immuncthempy. TRAIL Ie hekig considered as « novel tempeatic agent de ta tx lective cytotoxicity cancer
cals. Hover. presale eancer cl tines are resistant fo TRAM due, in large pat, the overexprestion of he
anf-apaptaie ge procact XIAP. The regulation af XEAP overexpression aod its trnscription and is roleia
regulating eaBlvily to TRAN hea boon investiga We exavined the PC-3 cal fice nea prototype model syste.
‘We hive found that ooasutive NF-L activity regulates the erste 10 TRAIL ox inhibition oF NICE sonstaed
the ells te TRAM -indaoed apoptosis, The inhibition by NI-La ws due in large prt to downregulation ofthe
_oti-apopttie gene products XIAP and Bele. Wie alsa deemastate thet the overerpession of Bela. and XIAP are
due tn larg parte the eoustintive activation of NELR by mmeor derived TNF-a in an sttoetine/pametine ante,
‘The ihiation of NE-KEYXEAPY BelxL and seridanon ty TRALL was coocoriaat with wpregulztion of DES. Fi.
bles, we demenstate that overstprestion of Bela. and XTAP een dered dhe eli resistant to CODP indeed
sportosis and ther ibibo sensiizs cells to CDDP apoptisa,

1 ALRP. spoptonie, ME kn, sensitization, Belmxt, ‘3

py; eytokinos, aruga 8 FRCE CODE

Urelasa: fied Unozassizied UUnclagsified muuinites
TEN Tea Fa ‘Mar a FT
‘TABLE OF CONTENTS

Cover

SF 29

‘Table of Contents

Introduetion-

Body-

[Key Resenreh Accomplishments —eee—_————-— nee ener nnn

References

Conclusions ~

‘Reportable Onteames -—-

Figures ~ —

Appendices (One publication and one abstract)
INTRODUCTION

“The filure o eradicate advanced prostate cancer that is resistant to conventional
shorapies, such as chemo and hormonal therapies, has Jed lo the exploration of navel
therapcutie epplications such a immunotherapy, One form of immunotherapy is to
generate anti-tumor cytotoxic mphocytes that can recognize and eradicate resistant
fumor cells. Cytotoxic lymphocytes mediate their killing by various mochuniams
including the perforin granzyme pathway and by members of the TNF-o. superfamily.
‘Among tho TNF-1 superfauily, TRAIL has been shown to be seloutively cytotoxic to
ceanccr cells el party cytotoxic to normal cells and is, chorofore, considered as a good
candidate for prostate cancer thonpy. However, the development of drug‘hormoxal
resistant prostate cancer resulls in the development of tumor colls rexistant co inunime
Leller eels and, indeed, prostato cancer cell Yines have boa shown to be relatively
resistin co TRAIL-indiced apoptosis, Resistanee is under the regulation of apoptotic
requlalory gene products in the cancer cells. We have demonstrated that prostate cancer
cells resistant co TRAIL wre due in large part to the evorexpression of The antiapoptotic
gene product, XTAP und Del-,., both af Which are under the regulation of eonstitulive
NEB, Inhibition of NF-KB or sithcr XIAP o Bel expression results in the reversal of
resistant turnor oells and sensitivity to TRAIL-inducod spoptorie. This proposal
investigates the role of XTAP in tesistanee to TRAIL and investigates the undsrlying
mechanisms of the regulation of XIAP expression in prostate eancsr cells. We have
proposed to investigate 1) The role of XIAP in protecting prostate emicer cells from
‘TRAIL-mediated apoptosis, 2) The regulation of XIAP by NF-rR and NF-<B repulation
bby XIAP, and 3) The role of endogenous TNF-<c amt IT-6 in the regulation of XIAP and
resistance of tumor cells to TRAIL-induced apoptosis
Bopy

Wo have made significant progress and novel findings related to the proposod
study during the second year.

41) Role of constintive NF-xP activity and downstream anii-apoptotic gene expression
(XIAP and Bel-y) in the tegutation of TRAIL resistance.

‘We have reported previonsly that prosivle luraor cells atc resistant to TRATL-
indueed apoptosis, The mechanism underlying resistance was explored and we have
reported the! overexpression of XIAP is involved in the resistance (Zisman ef af,
2001; Ng ef al, 2002). Wo and efhere have also demonstrated that prostate cancer cell
lines exhibit constitntvely active nuclear factor kappa B (NF-xB) (Suh et af, 202;
Hivorts-Yeper et al, 2004). NF-kB rogulstes the transcription of many anti-apoptotic
‘geuc producls including XIAP and Bel-y,. We examined the role and mechanism of
NF-xPtinduced resistance to TRAIL apoptosis. We used the aitric oxide donor
DETANONOate and the NF-xP inhihilior Bay 11.7085 to inhibit NF-xB activity,
and treated PC-3 cells resulted in downstream inhibition of both XIAP and Bots,
expression (Figure 1). The inhibition of NF-xB resulled in sensitization to TRAIL
spoptosis, Further, the role of Bol-y, in the regulation of TRALL resistance war
conoboruied by the uso of the chemical inhibitor 2amethoxyantinycin A which
sensitized PC-3 cells to TRATL-induced apoptosis, We further examined the
spoptotic-signaling pathways following treatment of PC-3 celle with the combination
of NP-eB inhibitors and TRAIC, sud demonstrate that the combination, but not single
agents alone, activate the milochondrial pathsvay and the activation of exipases 9 and
3 and the induction of apoplusis, The above findings have heen recently reported
(Hucria-Veper.ef af, 2004; Appendix 1).

2) Role of tumor-detived TNF-a in the constitutive activation of NE-KB in PC-3 cells
and regulation of TRAM. resistance.

‘We have previously reported that PC-3 cella aynthesize and secrete TNF-o, ad
‘INF is a resistant factor (Borselling ef af, 1995), Since TNF-cc is a major inducer
of NF-XB activity, we hypothesized that tumor-derived TNT-o1 may activate NIKE
jn PC- cells via an autocrinc! paracrine loop. The activation of NF-«B by TNF-.
will result in the activiatinn of the anti-apoptotic gene products regulated by NFB,
such 93 Bela, and XTAP. The exprcssion of these gene prodocts will then maintsin
the resistance of the tumor oclls fo TRAIL-induced apoptosis. This hypothesis was
tested experimentally and validated. We demonstrare that tantment of PC-3 cells with
exogenous TNF-a. activates NF-xB in PC cetls (Figure 2A). This finding was
‘confirmed by the use of the NFoxB inhibitor, Buy 11-7085 (Higure 28), The role on
‘erviogenous TNF-t in the rogulstion of NF-«B activity was demonstrated! in studies
in which th ePC-3 tumor cells were treated with recombinant soluble TNFRI
GINFR1} to neutealize the sceroted TNF-, und iabibits its signaling and activation
of NFkB, The finding demonstra thot such weatmont resulted in significant
inhibition of NT-xD activity (Figure 2C). Those findings suggested that Tica.
secreted by PC-3 eells play a major rote in the constitutive activation of NF-KB nl
2

4

resistant fo TRAIL via expression of XIAP and Belwy, Jn vivo prostate cancer colls
that do not socio TNF-o. may be influenced by TNF-t-derived fom the
microenvironment to regulate their resistance (o TRAIL-apoptosia, The above studies
anv lditional studios are being completed tor publication.

of DRS

Mechanism of NFcR mediated regulation of TRA. revistunee: Suppres
‘wanscription

‘We examined the potential mechanism by which NF-«8 activation, aside from the
expression of XIAP and Bcl-g, regulates PC-3 celle’ resistance to ‘TRAIL. We have
found that inhibiters of NF-KB, which induced sensitfzation of PC-3 to TRAT.
‘induced apoptosis, corrclstal with Ihe upregulation of the TRAIL receptor DRS. The
upregulation of DRS was determined by low, RT-PCR and westem (Figure 3). We
Then examined the upregulation of DRS expression using a luciferase reporter system
that we have obtained from our collaborator Dr. Sakai in Kyoto, Jepart (Yoshida et
ai, 2001), We analyrel constructs and demonstrate that NF-xB inhibitors such as
DHMEQ (Ariga ef af, 2002) significantly augmented Iuciferase activity and
confirmed the above findings (Figure 4), We ate currently examining several other
‘eonstructs of the reporter system devefoped by De. Sakal in order to identify NFA
zegutated trascription factor thal meviulate negatively the transcription of DRS, One
putative transcription fueloe is the tansceiption repressor Yin Yang 1 (VV1) that We
hyve previously reported to negatively regulate the teanseription of the Fas receptor
{Gorban and Bonavida, 2001),

Role of XIAP and Bey, expression in the regulation of BC.3 resistance to CDDP-
{induced apoptosis

‘We have Jound that prostate cancer tumor cell fines (PC-3, CL-1, LNCaP) are
sesistant to CDDP anediated apoptosis. We examined whether the resistance is duc in
part to the oonsftuive acvation of NF-xB and downstream regulaiion af XTAP and
Pela expression similar to the resistence obscrved against TRAIL. We also
Inypothesized that tumor-derived cytokines (e.g- TNF-a) that regulates the constitutive
activity of NF-«B and downstream anti-apoptotic geue products like XIAP and Bel
‘will result in the regulation of tumor eel resistance to CDDP. Hence, interfering with
Uhis pathway should sensitize the ostls to CDDP apoptosis (Figure 5). This hypothesis
‘ius tested and verified experimentally. We have found that inhibition of endogenous
"INE-c. by rcoomtbinat sTNFR| sensitizes PC-3 cells to CDDP-induced apoptosia
(Figure 5). Further, inhibition of NF-KB by Bay 11-7085 miwvicked the neutrafization
of TNT-a and sensitized the cells to CDDP apeptosis (Figure 6), As shown
previously in Figure I, the inhibition of NF-xB resulted jo the inhibition of XLAP and
Tel-. We demonstrate that inhibition of Bel. by the inhibitor 2-methoxyantimycin
A sensitizes oclls to CDDP-induced apoptosis. The dircet role of XJAP in the
inhibition of CTDP-induced apoptosis was examined by the use of actinomycin D
which we have earlier reported selectively inhibite XTAP expression (Ng and
Bonavida, 22), Troatment of PC-3 with Act D resulted in seusitization of PC-3
cells ta CDDP-induced apoptosis Figure 7). Those findings demonstrate thal NF-x Tb
and gene products XIAP and Bcl-a regulate the resistmce of PC-3 eells to CDDP-
induced apoptosis, These studies and others in progress will be completed for
By)

publication. The above preliminary findings were presented at a mini-aymposium it
‘the 2004 AACR meeting in Orlando, Florida A copy of the abstract is enclosod as
Appondix 2,

“Expression af XLAP in prostate tumor tissue microsmays

"We have completed the staining and specificity of XLAP expression in prostate
tumor tissue microarrays, Case materials from 246 prostatectomies were arrayed into
3 blocks encarapassing a total of 1,364 individual tissue cases, A standard 2-atep
indirec. aviin/biatin complex (ABC method was used), Included in the study YIN,
BPH, and normal tisruce rom the same pool were wnalyzed. In addition, the
Frequency of positive target cells (Range 0-100%4) will he scored for cach fissue
mietorrray spot, Stlistical analysis will examine the association betwoon XTAP
expression and clinica! pathological variables using the Pearson chi-syuare test.
Kaplso-Meler and Cox analyses will be performed by Dr. Steve Horvath, a
biosulistcian in the Department of Human Geneties, We anticipate the analysis to be
completed shorty.
REFERENCE:

‘Ariga A, Namekawa J, Matsumoto N, Inoue 1, Umczawa K. trihibition of tumor necrosis.
factorsipha -indnced nuclear translocation and activation of NF-kappa B by
detndroxymethylepoxyquinemicin, J Biol Cheen, 2002 Jul $;277(27}:24625-30, Epuh
2002 Apr 30,

Borselino N, Belldegrun A, Bonavida B, Endogenous interleukin 6 is a resistance factor
for cis-dismminedichloroplatinum and ctoposie-mediated cytotoxicity of human
‘prostate carcinoma cell Jines. Cancer Res. 1995 Oot 15;55(20):4633-9.

“Htorts-Yepes 8, Vega M, lazicchi A, Garban TT, Hongo F, Chong G, Bonavide B. Nitric
oxide sensitizes prostate carcinoma cell tines to TRAIL-mediated apoptosis via
inactivation of NF-kapps B and inhibision of Bel-xl expression. Oncogene. 2004 Jun
24;23(29):4993.5008,

‘Ng CP, Bonavids B, X-linked inhibitor of apoptosis (XIAP) blocks Apo? ligendtumor
necrosis faotorreleied apoptosissnducing ligand-mediated apoptosis of prosiate cancer
cells in the presence of mitochondrial activation: sensidzation by overexpression af
second mitochondria-derived aotivator of crypanetirect IAP-binding protein with low p!
(Sins0/DIABIO}, Mol Canocr Ther. 2002 Oct,}(12) 1051-2.

Ng CP, Zisman A, Bonavida B. Synergy is ackioved by comptementation with
Apo2LATRSIL and actinomycin D in Apo2L/TRATL-mediated apoptosis of prostale
cancer cells: role of XLAP in resistanoc, Prostate. 2002 Dec 1;53(4):286-99,

Shigeno M, Wakao K, Tehikawa T, Suzuki K, Kewakemi A, Abin S, Miyazoc S,
Nkagew ¥, lobikawa H, Hamasaki K, Nakata K, Whit N, Eguchi X. inerferomelpha
sensitizes human hepatoma ces to TRATE-induced apoptosis throngh DRS wpreguatian
and NI-kappa B inactivation. Oneayene. 13 Mar 20:22(11):1653-62,

Suh J, Payvandi E, Edelstein LC, Amenta PS, Zong WX, Gelinas C, Rabson AB.
Mechanisms of constitutive NF-appaB activation in human prostate cancer cells,
Prostate, 2002 Aug 1;52(3}183-200,

‘Villnan DM, Wraradjene K, Szues KS, Douglas 1, Houghton JA, Rottlerin sensilizes
‘colin carcinoma eels to tumor ncorosis faclor-related apoptosis-inducing ligant-induced
spoptosis via unccupling of the mitochondria independent of provein kinase C. Cauoer
‘Rea, 2003 Aug 15;63(16):5118-25.

Yoshie T, Maeda A, Tani N, Sakai T. Promoter structure and transcription
sites of he human death receptor S/TRAT-R? yene. FEBS Lett, 2001 Nov 2;507(3):381+
5.

Zisman A, Ne CB, Pantuck AJ, Bonovida B, Belldegrm AS, Actinomycin D and
(gemcitabine synergistically sensitize androgen-independent prostate cancer calls to
‘Apo2T/TRAT-mediated apoptosis. I Immunother. 2001 Nov-Deo;34(6):459-71,
‘KEY RESEARCH ACCOMFLISHMENT!

We have completed studies on the expression of XLAP on prostare cancer tissus
snieronrrays aril analyses are curently beiny performed

We have demonstrated that proslate eancor cll lines express constitutively, wetivated
NEB, NF-KB regulates the anscription of XIAP ond Bels and inhibition of NF
k downreyulates their expression anil sensitizes the colls to TRATL-indnecd
apoptosis.

We have demoustrated thal PC-3 cells scorcte TNF-r which acts by
antoctinelparserine Suhion as an activator of NE-xB. Inhibition of TNF-a by anti-
TINF-x antibody or by soluble TNFR downegulates NF-xB activity and the
expression of XIAP and Bel-a,and sensitizes the ells o TRAIL-induced apoptosis

We have demonstrated that NF-KB negatively regulates the expression of the TRAIL
receptor, DRS. Inhibition of NI-eD by chemical inhibitors or by nitvic oxide donor
(Grhich Snuitrosylates p50) resulted in the upregulation of DRS expression and
sensitization to TRAIL-induced apoptosis, These findings were conuborated in POS
cells ttasfected with the DRS promoter and treatment with NF-cB inhibitors or by
nitric oxide resulted insignificant increase in the tucifrase activity.

‘We have also found that NF-xB regulates Bel, expression in PC-3 calls, Inhibition
of Bol, sefivily results in sensitization of the calls to TRALL-mediated apoptosis,
‘Those findings reveal that in PC-3, bouk XIAP and Bek, overexpression regulates
the sensitivity of prostate cancer ells to TRAIL apoptosis,

‘We have found thi NF-xB also regulates BelL expression jn prostate cancer cclls
and both XTAP and Bcluy overexpression render the cells resistant to
chomothenspentic deugs-inducod apoptosis, This finding suggests that there ia crose-
resistance herween inmuno-mediatod and drug-induced apoptosis,
REFEI 8 AND APPENDICES

1. Huerts-Yepea S, Vege M, Jazirchi A, Garban H, Hougo F, Cheng G, and Bonsvida B.
Nittie oxide sensitizes prostate earcinoma cell lines i‘ TRATL-mediated apuptosis via
inactivation of NF-kB anu) inhibition af Bcl-xL expression. Oncogene, 23: 4993+
3003, 2004

2, Hucrta-Yepez 8, Vega M, Hongo F, Jazirehi A, Garbum H, Mizutani ¥, nd Bonavida
B. Regulation of proslate eanver sensitivity to apoptosis by CODP by dovmregulation
of SIAP and Bel-y, expression via iuhbition of consltulive NPoxB acsvity. Abstract
No, 4826, AACR March 2004 Annual Mcoting- Manuscript in Frcparation

3. Huesta-Yopez S, Vega M, Escoto-Chavez SB, Muntock B, Sakai T, and Bonavida B.

lnhibition of DRS expression and rogulalion of TRAIL resistance in cancer cells by
the transcription repressor YY. CMisnuseript in preparation),

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