DTIC ADA593709: Regulation of TRAIL-Medicated Apoptosis in Prostate Cancer by Overexpression of XIAP

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AD_________________

Award Number: DAMD17-02-1-0023

TITLE: Regulation of TRAIL-Medicated Apoptosis in Prostate Cancer by Overexpression
of XIAP

PRINCIPAL INVESTIGATOR: Benjamin Bonavida, Ph.D.

CONTRACTING ORGANIZATION: The University of California
Los Angeles, CA 90024-1406

REPORT DATE: January 2006

TYPE OF REPORT: Final

PREPARED FOR: U.S. Army Medical Research and Materiel Command
Fort Detrick, Maryland 21702-5012

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4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
Regulation of TRAIL-Medicated Apoptosis in Prostate Cancer by Overexpression
of XIAP 5b. GRANT NUMBER
DAMD17-02-1-0023
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6. AUTHOR(S) 5d. PROJECT NUMBER
Benjamin Bonavida, Ph.D.
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E-mail: [email protected]
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The University of California
Los Angeles, CA 90024-1406
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U.S. Army Medical Research and Materiel Command
Fort Detrick, Maryland 21702-5012
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Approved for Public Release; Distribution Unlimited
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14. ABSTRACT
Patients with prostate cancer (CaP) develop resistance to conventional therapies and
alternative therapies, such as immunotherapy, are being actively considered. TRAIL is
selectively cytotoxic to tumor cells and minimally cytotoxic to normal tissues and is a
candidate for immunotherapy. CaP cells, however, are resistant to TRAIL due to antiapoptotic
mechanisms such as overexpression of XIAP. This proposal investigated the
mechanism by which XIAP regulates resistance to TRAIL and the findings demonstrate that
TRAIL resistance is regulated by the expression of the TRAIL receptor DR5. The regulation
of DR5 was found to be under the control of the transcription repressor YY1. Inhibition of
NF-κB inhibited both YY1 and XIAP and sensitized the cells with TRAIL-induced apoptosis.
We demonstrate that YY1 regulates the transcription of DR5 via a YY1 DNA-binding site on
the DR5 promoter. The clinical importance of XIAP expression in prostate cancer tissue was
examined in tissue microarrays and we demonstrate its prognostic significance in prostate
cancer patients. This also correlated with the overexpression of YY1 which also showed its
prognostic significance in prostate cancer patients. In addition, we have demonstrated
that cytokines derived from prostate cancer and/or from the tumor microenvironment
regulate the constitutive activation of NF-κB and downstream XIAP and YY1 and regulate
resistance to TRAIL. Overall, our findings identify new therapeutic targets for TRAIL.

15. SUBJECT TERMS
TRAIL, signaling, XIAP, apoptosis, NF-κB, sensitization, Bcl-xL, Immunotherapy, cytokines, drugs

16. SECURITY CLASSIFICATION OF: 17. LIMITATION 18. NUMBER 19a. NAME OF RESPONSIBLE PERSON
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U U U UU 363 code)

Standard Form 298 (Rev. 8-98)
Prescribed by ANSI Std. Z39.18
Table of Contents

Introduction…………………………………………………………….…………....4

Body…………………………………………………………………………………….6

Key Research Accomplishments………………………………………….………14

Reportable Outcomes……………………………………………………………….16

Conclusions…………………………………………………………………………..19

References……………………………………………………………………………20

Appendices……………………………………………………………………………23
Introduction

Prostate cancer (CaP) is the most common malignancy and age-related cause of cancer
deaths in American males. Treatments for metastatic prostate cancer include hormonal ablation,
chemotherapy, and combination therapies. These treatments are aimed at inhibiting tumor
growth and at also inducing apoptosis. Unfortunately, hormonal therapy is always followed by
the relapse of an aggressive androgen-independent disease that is insensitive to further hormonal
manipulation or to treatment with conventional chemotherapeutic drugs. The underlying
mechanisms by which the prostate tumor cells develop resistance to hormonal/drug-mediated
effects are poorly understood. It is probable that the tumor cells acquire mechanisms that resist
androgen ablation/drug-mediated apoptosis. Failure to eradicate advanced resistant tumors with
conventional therapies has led to the exploration of novel therapeutic approaches such as
immunotherapy.
Immunotherapy is generally aimed at the generation of anti-tumor cytotoxic lymphocytes
that can recognize and eradicate the drug-resistant tumor cells. Also, immunotherapy is
predicated on the notion that all drug-resistant tumors should succumb to cytotoxic lymphocyte-
mediated killing. Tumors that develop anti-apoptotic mechanisms to escape drugs/radiation-
mediated apoptosis may also develop cross-resistance to apoptosis mediated by cytotoxic
lymphocytes. We and others have reported that many types of drug-resistant tumor cells
including CaP cells are also resistant to immune-related cytotoxic mechanism of killing. Thus,
the development of immune resistance by drug-refractory tumors may hinder the therapeutic
effect of immunotherapy. However, if the tumor can be sensitized to overcome resistance, the
combination of sensitization and immunotherapy may be more efficacious. The underlying
molecular mechanisms of sensitization to immunotherapy are therefore important to explore for
the identification of gene targets involved in resistance and hence develop new means to modify
these targets and reversing immune resistance.
The inhibitor of apoptosis proteins (IAPs) represent a family of endogenous caspase
inhibitors that share a conserved structure known as the BIR domains (Reed, 2001). Eight
encoding IAPs genes are found in the human genome, and some of these are overexpressed in
cancers (Ferreira et al., 2001; Hofmann et al., 2002; Tamm et al., 2000). These proteins are
involved in maintaining tumor cell survival or for regulating resistance to apoptosis induction by
various therapeutics (Bilim et al., 2003). The X-chromosome linked IAP (XIAP) is the best
characterized of the IAP family members in terms of its caspase inhibitory mechanism. The
XIAP protein contains three BIR domains. XIAP levels have been shown to be pathologically
elevated in many acute and chronic leukemia, prostate cancer, lung cancer, and other tumor
(Byrd et al., 2002; Ferreira et al., 2001; Hofmann et al., 2002; Schimmer et al., 2003; Seligson et
al., 2006). Schimmer et al., (2004) have recently reported that small molecule antagonists of
apoptosis suppress XIAP function and exhibit a broad anti-tumor activity and such molecules are
considered as targets for cancer therapy.
The TNF ligand superfamily serves as an important role in the host immune defenses
against cancer as an inducer of apoptosis in tumor cells. Apo2L/TRAIL recently has drawn
interest as a potential effective anti-tumor therapeutic agent in a variety of cell lines since it is
selectively cytotoxic against transformed cells and not against the majority of normal cells. Our
recent findings have demonstrated that drug-resistant CaP cells are resistant to TRAIL and also
demonstrated that chemotherapeutic drugs (e.g. Act D, ADR and VP-16) sensitize CaP cells to
TRAIL-mediated apoptosis (Zisman et al, 2001; Ng et al., 2002). Unlike a recent study showing
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FLIP overexpression is responsible for TRAIL (Fulda et al., 2000), we have also identified a
member of inhibitor of apoptosis proteins (IAPs), XIAP, as responsible for resistance in CaP.
Therefore, this project is designed to investigate the role and regulation of XIAP overexpression
in CaP in order to find new means to inhibit its expression by new agents and reverse TRAIL
resistance for clinical use.
Our proposed studies are relevant to the success of CaP immunotherapy. Accumulating
evidence suggest overexpression of IAPs, particularly XIAP, is a cause of apoptosis dysfunction
in cancer cells (Holcik et al., 2000; Kitada et al., 2000). For instance, Tamm et al. (2000)
reported the relevance of XIAP in vivo responses to cytarabine in AML. Patients with lower
XIAP protein had significantly longer survival and a tendency towards longer remission duration
than those with higher levels of XIAP. In prostate cancer, we identified XIAP as and important
anti-apoptotic gene product that regulates TRAIL apoptosis.
In this grant application, we have proposed to investigate the following aims: 1) The role
of XIAP in protecting CaP cells from TRAIL-mediated-apoptosis. Examination of the direct role
of XIAP by transfection utilizing the newly reported IAP inhibitor Smac/DIABLO and by XIAP-
antisense. The sensitivity to TRAIL and signaling pathway for apoptosis in transfectants and
normal CaP will be compared. Further, we proposed to correlate the expression of both XIAP
and Smac/ DIABLO in freshly derived normal, benign, and human prostate tumor cells at
different stages of the disease and establish correlations with prognosis. 2) The role of
constitutively activated NFκB (survival factor) in the regulation of both resistance to TRAIL and
XIAP-expression. We proposed to examine the role of NFκB in the regulation of XIAP
expression and TRAIL sensitivity. 3) The roles of constitutive and exogenous TNF-α and IL-6
in the regulation of NFκB, XIAP expression, and sensitivity to TRAIL.

5
Body

The following tasks were proposed for investigation:

Task 1: The role of XIAP in protecting CaP cells from TRAIL-mediated apoptosis
Task 2: Regulation of XIAP by NFκB and NFκB regulation of XIAP
Task 3: To determine the role of endogenous TNF-α and IL-6 in the regulation of XIAP and
resistance to TRAIL
We have investigated all of the above proposed tasks and several reports emanated from
our findings. Further, we have made several novel findings and have identified new gene
products that regulate TRAIL sensitivity in prostate cancer cells. The following summarizes our
progress to date.
We have proposed to examine in Task 1 the direct role of XIAP in the regulation of
TRAIL resistance by transfection experiments utilizing the newly reported IAP inhibitor,
Smac/DIABLO and by XIAP antisense. We have also proposed to examine the sensitivity and
signaling to TRAIL in the transfectants. We have completed the studies with the transfection of
CaP cells with Smac/DIABLO and the studies have been reported in two publications (Ng, et al.
2002; Ng and Bonavida, 2002a). Briefly, the studies provided novel findings in the mechanism
of XIAP regulation of TRAIL resistance. Noteworthy, we show in CaP cells that TRAIL
signaling results in the loss of mitochondrial potential, the release of cytochrome c and
Smac/DIABLO into the cytoplasm in the absence of caspase activation due to overexpression of
XIAP. However, inhibition of XIAP by transfection with Smac/DIABLO (or treatment with
Actinomycin D, which inhibits XIAP expression) allowed the TRAIL signaling to proceed to
activation of caspase 9 and 3 and induction of apoptosis. These findings demonstrated that two
complementary signals are involved to overcome prostate cancer as well as possibly other
cancers to TRAIL-induced apoptosis. The two signal model has been proposed in the studies that
we have reported and has also been detailed in a recent review that was published in Advances in
Cancer Research (Ng and Bonavida, 2002b).
In Task 1 we have also proposed to examine the expression of XIAP and Smac/DIABLO
in freshly derived normal, benign, and prostate tumor cells at different stages and grades and
correlation with prognosis. We have examined the expression of Smac/DIABLO in tumor cell
lines by immunohistochemistry (IHC). We have established the optimal conditions and
specificity of the antibody against Smac/DIABLO and demonstrated successfully that the
antibody is applicable for IHC in addition to its usage for Western blot in our publications.
Due to limitations in CaP tissue microarrays, we first examined the clinical role of XIAP
overexpresssion in such microarrrays.

Prognostic significance of XIAP expression in CaP tissue microarrays
Our findings have established that overexpression of XIAP in CaP is associated with both
chemoresistance and immune resistance to TRAIL-induced apoptosis. We hypothesize that
overexpression of XIAP in human CaP tissues may be associated with resistance and thus, may
be of prognostic clinical significance. We examined the expression of XIAP by
immunohistochemistry using CaP tissue microarrays and the data were statistically analyzed.
The findings demonstrate that overexpression of XIAP is of prognostic significance in a subset
of patients with prostate cancer. A manuscript has been completed and will be submitted shortly
(Seligson et al., 2006; appendix). The abstract is highlighted below.
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Abstract
Objective: The X-linked Inhibitor of Apoptosis (XIAP), a member of the family proteins, has been
linked to tumor cell survival and drug resistance by direct blockade of caspase-mediated
extrinsic apoptotic pathways. Thus, XIAP status may help predict prostate cancer recurrence
and clinical response to therapies relying on unencumbered apoptotic machinery. It is therefore
important to validate the foundational protein expression patterns of XIAP and examine its
prognostic implications in human prostate cancer.
Methods: Immunohistochemistry was performed on tissue microarrays constructed from paraffin
embedded primary prostate cancer specimens from 226 hormone naïve patients who underwent
radical retropubic prostatectomy. 223 cases provided informative epithelium for XIAP analysis
encompassing 1,107 total tissue microarray spots including morphologically normal prostate
(NL; n=252), benign prostatic hyperplasia (BPH; n=122), prostatic intraepithelial neoplasia
(PIN; n=48) and invasive prostate cancer (Cancer; n=685). XIAP expression was scored in a
semi-quantitative fashion using an integrated intensity measure (0.0-3.0). The protein expression
distribution was examined across the spectrum of epithelial tissues and its association with
standard clinicopathological covariates and tumor recurrence was examined in 192 outcome-
informative patients.
Results: The mean XIAP expression was significantly higher in prostate cancer (intensity = 1.32)
compared to PIN (intensity = 1.08; p=0.019), normal (intensity = 0.78; p<0.0001), and BPH
(intensity = 0.57; p<0.0001). 69% of BPH stained negatively to weakly (intensity <1.0), 53% of
normal, 37% of PIN and only 26% of prostate carcinomas. With XIAP expression dichotomized
at an intensity of 1.8, XIAP is an independent predictor of tumor recurrence in multivariate Cox
proportional hazards analysis in all patients (P = 0.0025; HR = 8.92; 95% CI = 2.16-38.86), as
well as after substratifying by Gleason score (P = 0.010; HR = 6.61; 95% CI = 1.57-27.89 for
high Gleason score [7-10] cases). In patient substrata with low Gleason score tumors [2-6], no
patients (0%) with an XIAP intensity > 1.8 (n=23) experienced tumor recurrence, while 26%
with low XIAP (n=89) recurred. Patients with high grade or non-organ confined tumors with
high XIAP have a lower risk of recurrence as a group than any patients whose tumors express
low XIAP, even those of low grade or that are organ confined. These data are consistent with
findings in a recent report (Krajewska 2003).
Conclusions: XIAP is expressed at higher levels in prostate cancers compared to matched
normal tissues. High XIAP expression is strongly associated with a reduced risk of tumor
recurrence, and is not directly associated with Gleason score, tumor stage, capsular involvement
or preoperative PSA status, suggesting that it is a novel prognosticator and a potential target for
prostate cancer diagnosis and therapy. Based on these results, patients with lower XIAP
expression in tumors are most in need of therapeutic intervention and may also be most
responsiveness to chemotherapeutic and death receptor targeted therapies.

In task 2 and 3 we have proposed to investigate the role of NF-κB and TNF-α in the regulation
of XIAP.

1. Role of constitutive NF-κB activity and downstream anti-apoptotic gene expression (XIAP and
Bcl- ) in the regulation of TRAIL resistance.
xL
We and others have also demonstrated that prostate cancer cell lines exhibit constitutively active
nuclear factor kappa B (NF-κB) (Suh et al., 2002; Huerta-Yepez et al., 2004). NF-κB regulates
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the transcription of many anti-apoptotic gene products, including XIAP and Bcl- . We
xL
examined the role and mechanism of NF-κB-induced resistance to TRAIL apoptosis. We used
the nitric oxide donor DETANONOate and the NF-κB inhibitior Bay 11-7085 to inhibit NF-κB
activity, and treated PC-3 cells resulted in downstream inhibition of both XIAP and Bcl-
xL
expression. The inhibition of NF-κB resulted in sensitization to TRAIL apoptosis. Further, the
role of Bcl- in the regulation of TRAIL resistance was corroborated by the use of the chemical
xL
inhibitor 2-methoxyantimycin A which sensitized PC-3 cells to TRAIL-induced apoptosis. We
further examined the apoptotic-signaling pathways following treatment of PC-3 cells with the
combination of NF-κB inhibitors and TRAIL, and demonstrate that the combination, but not
single agents alone, activate the mitochondrial pathway and the activation of caspases 9 and 3
and the induction of apoptosis. The above findings have been recently reported (Huerta-Yepez et
al., 2004).

2. Regulation of prostate carcinoma cell line resistance to TRAIL via upregulation of DR5
expression.
We have previously reported that treatment of CaP with nitric oxide donors or inhibitors
of NF-κB resulted in sensitization to TRAIL-induced apoptosis (Huerta-Yepez et al., 2004). We
examined the mechanism of resistance by delineating putative transcription factors, aside from
NF-κB, that may be involved in DR5 transcription. We demonstrate, by using various constructs
of the DR5 promoter, that deletion in the promoter of a region with a putative YY1 DNA binding
site resulted in significant augmentation of luciferase activity and suggested that YY1 may
negatively regulate DR5 transcription. This finding was corroborated by demonstrating that
mutation in the DNA binding site also resulted in augmentation of luciferase activity. In addition,
inhibition of YY1 via chemical modification with nitric oxide or by transfection with sIRNA for
YY1 resulted in both upregulation of DR5 expression and sensitization to TRAIL-induced
apoptosis. Altogether, these findings demonstrate that inhibition of NF-κB, which results in
downstream inhibition of YY1 and XIAP, results in inhibition of YY1 activity, leading to
upregulation of DR5 and subsequent sensitization to TRAIL. The findings are in a manuscript
that will soon be submitted for publication (Huerta-Yepez et al., 2006). The abstract is
highlighted below.

Abstract
Most tumors are resistant to TRAIL and need to be sensitized to undergo apoptosis. We
have recently reported that TRAIL-resistant human prostate carcinoma cell lines can be
sensitized by various NF-κB inhibitors (Huerta-Yepez et al., 2004), and sensitization correlated
with upregulation of DR5 expression. We hypothesized that a gene product(s) regulated by NF-
κB with DR5 repressor activity may be responsible for the DR5 regulation. Inhibition of NF-κB
activity resulted in significant upregulation of DR5 expression and sensitized prostate tumor
cells to TRAIL-mediated apoptosis and synergy is achieved. Treatment of PC-3 cells with NO
inhibited both NF-κB and YY1 DNA-binding activity and also inhibited YY1 expression.
Treatment of PC-3 cells with YY1 siRNA resulted in upregulation of DR5 expression and
sensitization to TRAIL-induced apoptosis. The direct role of YY1 in the regulation of DR5
expression was examined in an DR5 luciferase reporter system (pDR5). Two constructs were
generated, the pDR5/-605 construct with a deletion in the promoter region containing the
putative YY1 DNA-binding region (-1224 to -605) and a construct pDR5-YY1 with a mutation of
the YY1 DNA-binding site. Transfection of PC-3 cells with these two constructs resulted in
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significant (3-fold) augmentation of luciferase activity over baseline suggesting the repressor
activity of YY1. The present findings demonstrate that YY1 negatively regulates DR5
transcription and expression and hence, regulates resistance to TRAIL-induced apoptosis.
Inhibitors of YY1 expression and/or activity in combination with TRAIL may be useful in the
treatment of TRAIL-resistant tumor cells.

3. Chemical modification of the transcription repressor YY1 by nitric oxide: mechanism of NO-
induced upregulation of DR5 and sensitization to TRAIL-induced apoptosis
The above findings in section 1 (above) and 3 (below) have clearly demonstrated that
DR5 transcription is negatively regulated by the overexpression of the transcription repressor
YY1 in CaP and overexpression of YY1 is regulated by the constitutively activated NFkB
activity which also regulates XIAP. In addition to the transcription regulation of YY1 by NO
donors and by drugs, we demonstrate that NO also directly modifies YY1 via S-nitrosylation and
thus, preventing its DNA binding activity and transcription DR5 repressor activity. These studies
have been recently published (Hongo et al., 2005) and the abstract is presented below.

Abstract
Treatment of several prostate cancer (CaP) cell lines (PC-3, CL-1, and DU-145) with the
nitric oxide (NO) donor DETA/NONOate upregulated Fas expression and sensitized the CaP
cells to the Fas ligand CH-11 agonist monoclonal antibody-induced apoptosis. Previous findings
demonstrated that the transcription repressor Yin Yang 1 (YY1), which is inhibited by NO,
negatively regulates Fas transcription [H.J. Garban, B. Bonavida, Nitric oxide inhibits the
transcription repressor Yin-Yang 1 binding activity at the silencer region of the Fas promoter: a
pivotal role for nitric oxide in the upregulation of Fas gene expression in human tumor cells, J.
Immunol. 167 (2001) 75-81]. YY1 is a zinc finger protein and thus, we hypothesized that NO
inhibits YY1 activity via S-nitrosation of critical cysteines residues coordinated by Zn2+.
Treatment of PC-3 cells with DETA/NONOate inhibited the constitutive DNA-binding activity of
YY1 as assessed by EMSA. Further, treatment with DETA/NONOate resulted in S-nitrosation of
YY1 as detected by two different methods. The DAN-based method examined NO-treated tumor-
derived cell lysates that were immunoprecipitated with an anti-YY1 specific antibody and the NO
released was determined quantitatively by fluorometry. The second method consisted of
immunoprecipitation of the tumor cell lysates by an anti-SNO cysteine antibody and the
immunoprecipitate was immunoblotted with anti-YY1 antibody. Both methods revealed
significant S-nitrosation of YY1 by DETA/NONOate treatment over control untreated cells. The
S-nitrosation of YY1 was further corroborated by immunohistochemistry using dual color
immunofluorescence. The direct role of YY1 in the negative regulation of Fas expression was
demonstrated by transfection of cells with siRNA YY1. The transfectants exhibited upregulation
of Fas expression in the absence of treatment with DETA/NONOate and were sensitized to CH-
11-induced apoptosis. Altogether, these findings reveal that NO inhibits YY1 DNA-binding
activity through S-nitrosation and consequently results in upregulation of Fas expression and
tumor cell sensitization to Fas-induced apoptosis.

4. Role of chemotherapeutic drugs in the sensitization of CaP to TRAIL-induced apoptosis
Previous studies have reported that treatment of tumor cell lines with chemotherapeutic
drugs such as adriamycin or CDDP resulted in upregulation of DR5 expression and sensitization
to TRAIL-induced apoptosis. The mechanism underlying the drug-induced regulation of DR5
9
expression is not known. Our studies with the chemotherapeutic drugs corroborate the above
findings. In addition, we demonstrate that treatment of CaP with CDDP resulted in inhibition of
NF-κB and YY1 activities, resulting in upregulation of DR5. We demonstrate that inhibitors of
XIAP, NF-κB and YY1 mimic CDDP-induced effects. In addition, we demonstrate that YY1 is a
transcription repressor and negatively regulates DR5 expression and thus, its inhibition by CDDP,
reverses the repression and upregulates DR5 transcription and sensitizes CaP to TRAIL-induced
apoptosis. Further studies, as demonstrated above for NO-mediated effects using reporter
systems, confirmed the role of CDDP-induced inhibition of YY1 in the regulation of DR5
expression and sensitivity to TRAIL. A manuscript has been prepared and will soon be submitted
for publication (Baritaki et al., 2006; Appendix). The abstract of this publication in highlighted
below.

Abstract
Cancer patients initially respond to treatment with chemotherapy, however, recurrences
occur and the tumors become refractory to further chemotherapy. Immunotherapy is currently
being investigated as an alternative to overcome drug resistance. TRAIL, a member of the TNF
family, has been shown to kill sensitive tumor cells with minimal toxicity to normal tissues and is
a new candidate for immunotherapy. Many drug-resistant tumor cells are also resistant to
TRAIL and such tumors require sensitization to reverse TRAIL resistance. We and others have
reported that several sensitizing agents (ex. VP-16, CDDP, ADR, chemical inhibitors, etc.) in
combination with TRAIL result in reversal of resistance to TRAIL apoptosis. Sensitization
correlated with the upregulation of DR5 expression. This study examined the mechanism
underlying the upregulation of DR5 expression. We hypothesize that the sensitizing agents may
inhibit a transcription repressor acting at the DR5 promoter. Treatment of drug resistant PC-3
tumor cells with drugs (example CDDP, vp-16, adriamycin, vincristine) sensitized the tumor
cells to TRAIL-induced apoptosis and apoptosis correlated with upregulation of DR5 expression
and inhibition of YY1. By examining the promoter of DR5, we detected the presence of one
putative binding site for the transcription repressor YY1. We examined whether YY1 negatively
regulates DR5 transcription and whether YY1 inhibition by the drug upregulates DR5 expression.
We used PC-3 cells transfected with a luciferase reporter system (pDR5 WT) and plasmids in
which the YY1 binding site was either deleted (pDR5 -605) and/or mutated (pDR5/YY1 mutant).
The findings revealed that the baseline reporter activity was significantly augmented in cells
transfected with either the deleted or mutated plasmids. In addition, CDDP treatment augmented
the luciferase activity in the WT reporter system, whereas there was no augmentation in the
deleted or mutant transfected cells. The direct role of YY1 in the upregulation of DR5 expression
and sensitization to TRAIL-induced apoptosis was demonstrated in cells treated with siRNA YY1.
The findings demonstrate that drug-induced upregulation of DR5 and sensitization to TRAIL is
mediated through inhibition of the transcription repressor YY1. Inhibition of YY1 correlated with
sensitization to TRAIL-induced apoptosis.

5. Role of XIAP and Bcl- expression in the regulation of PC-3 resistance to CDDP-induced
xL
apoptosis
We have found that prostate cancer tumor cell lines (PC-3, CL-1, LNCaP) are resistant to
CDDP-mediated apoptosis. We examined whether the resistance is due in part to the constitutive
activation of NF-κB and downstream regulation of XIAP and Bcl- expression similar to the
xL
resistance observed against TRAIL. We also hypothesized that tumor-derived cytokines (e.g.
10