Group Title: BMC Cancer
Title: Molecular fingerprinting of radiation resistant tumors: Can we apprehend and rehabilitate the suspects?
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Title: Molecular fingerprinting of radiation resistant tumors: Can we apprehend and rehabilitate the suspects?
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Creator: Rosser,Charles
Gaar,Micah
Porvasnik,Stacy
Publication Date: 2009
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Abstract: Radiation therapy continues to be one of the more popular treatment options for localized prostate cancer. One major obstacle to radiation therapy is that there is a limit to the amount of radiation that can be safely delivered to the target organ. Emerging evidence suggests that therapeutic agents targeting specific molecules might be combined with radiation therapy for more effective treatment of tumors. Recent studies suggest that modulation of these molecules by a variety of mechanisms (e.g., gene therapy, antisense oligonucleotides, small interfering RNA) may enhance the efficacy of radiation therapy by modifying the activity of key cell proliferation and survival pathways such as those controlled by Bcl-2, p53, Akt/PTEN and cyclooxygenase-2. In this article, we summarize the findings of recent investigations of radiosensitizing agents in the treatment of prostate cancer.
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BioMed Central


Review

Molecular fingerprinting of radiation resistant tumors: Can we
apprehend and rehabilitate the suspects?
Charles J Rosser*, Micah Gaar and Stacy Porvasnik


Address: Department of Urology, The University of Florida, Gainesville, Florida, 32610, USA
Email: Charles J Rosser* charles.rosser@urology.ufl.edu; Micah Gaar mgaar@fsu.edu; Stacy Porvasnik stacy.porvasnik@ufl.edu
* Corresponding author


Published: 9 July 2009
BMC Cancer 2009, 9:225 doi:10.1 186/1471-2407-9-225


Received: 3 February 2009
Accepted: 9 July 2009


This article is available from: http://www.biomedcentral.com/1471-2407/9/225
2009 Rosser et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Radiation therapy continues to be one of the more popular treatment options for localized
prostate cancer. One major obstacle to radiation therapy is that there is a limit to the amount of
radiation that can be safely delivered to the target organ. Emerging evidence suggests that
therapeutic agents targeting specific molecules might be combined with radiation therapy for more
effective treatment of tumors. Recent studies suggest that modulation of these molecules by a
variety of mechanisms (e.g., gene therapy, antisense oligonucleotides, small interfering RNA) may
enhance the efficacy of radiation therapy by modifying the activity of key cell proliferation and
survival pathways such as those controlled by Bcl-2, p53, Akt/PTEN and cyclooxygenase-2. In this
article, we summarize the findings of recent investigations of radiosensitizing agents in the
treatment of prostate cancer.


Review
Radiotherapy as a single modality has a limited but
important role in the overall treatment of solid tumors,
specifically prostate cancer. One major difficulty with
radiation therapy is that there is a limit to the amount of
radiation that can be safely delivered to the target organ
[1,2]. For prostate cancer, radiation doses are generally
limited to < 80 Gy because of the increased risk of toxicity
at higher doses and the lack of clinical evidence that doses
> 80 Gy improve local tumor control. Unfortunately, at
these dosing levels a significant proportion of tumors are
resistant to radiotherapy either they do not respond to
radiotherapy or they recur after treatment. It has become
clear that the resistance of human cancers to radiation cor-
relates with the expression of several key genes that regu-
late different steps of the apoptotic and cell cycle pathway.
Thus, the strategy of targeting distinct molecular pathways
may translate into higher efficacy, resulting in better sur-


vival. Radiation therapy combined with these targeted
therapies may exert enhanced antitumor activity through
synergic action. The combination treatment could also
decrease the toxicity caused by radiation therapy if lower
doses could be employed. In this article, we review the
current literature on key molecules that have been associ-
ated with the development of radiation resistant prostate
cancer and discuss novel ways to modulate these mole-
cules to sensitize these tumors to the killing effects of radi-
ation.

In the clinical arena, disease relapse after definitive radia-
tion therapy (i.e., brachytherapy, external beam radiation
therapy and proton therapy) is usually identified by an
elevated or rising prostate-specific antigen (PSA) profile.
Due to the controversy that surrounds identifying disease
relapse, a consensus panel was formed to define radiation
failure [3]. In addition to the debate about defining fail-


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ure, controversy exists as to whether this new disease is
persistent disease that was inadequately treated or recur-
rent disease that is refractory to therapy. It is difficult to
distinguish persistent disease from recurrent disease. It is
possible that persistent disease may have a genotype and
phenotype different to recurrent disease since the organ
has been exposed to radiation, albeit subtherapeutic
doses.

Depending on the initial serum PSA level, Gleason score
on biopsy, or clinical stage, 33-85% of patients undergo-
ing external beam radiotherapy (XRT) for localized pros-
tate cancer are biochemically disease-free 5 years after
initial radiation therapy [1,2,4-8]. Tumor response to
radiation is dependent on the total dose of radiation that
can be safely delivered. Prostate tumors are notoriously
resistant to total doses of 75 to 80 Gy, but higher doses are
not generally administered because of the increased risk of
toxicity. Because improved local tumor control is a thera-
peutic goal for most cancers, various strategies for sensitiz-
ing tumors to radiation have been tested over the last 20
years [9-13]. All of these strategies have involved the sys-
temic administration of drugs or other agents that have
specific toxicities of their own, which almost always limits
pharmacological doses to levels below what is needed to
sensitize tumors. The only successful sensitizing strategy
thus far has been hormonal deprivation in combination
with radiation therapy, which has been demonstrated to
improve cause-specific survival in men with advanced
prostate cancer [14,15 but still hormonal therapy in this
setting is not the panacea. Perhaps molecular targets iden-
tified in in vitro and in vivo work could help improve the
efficacy of radiotherapy above and beyond what we see
with hormonal therapy. Despite ongoing clinical trials, no
sensitizing strategies are currently available for wide-
spread use.


Table I: Genes associated with radiation resistant prostate cancer


Major


Various genetic abnormalities have been associated with
radiation-resistant prostate cancer [16]. Table 1 lists some
of the more common genetic abnormalities associated
with prostatic disease relapse after definitive radiation
therapy [17-25]. The four most common genetic abnor-
malities reported in the literature, p53, Bcl-2, COX and
Akt/PTEN will be reviewed in detail.

PI3KIAktIPTEN
PI3K is a lipid kinase that can generate phosphatidylinosi-
tol-3,4,5-trisphosphate (PI(3,4,5)P3) which has a broad
array of functions including inositol phosphate metabo-
lism, phosphatidylinositol signaling system, p53 signal-
ing pathway, cellular proliferation and survival, focal
adhesion, and cell-cell communication. PI3K is regulated
by its upstream growth factor receptor tyrosine kinases
(e.g., EGFR family receptors). In addition, there is cross
talk between the PI3K/Akt pathway and mitogen-acti-
vated protein kinases (MAP Kinase) pathway, mTOR
pathway, and protein kinase A, B and C (PKA, PKB, PKC)
pathways [26-29]. Phosphatase and tension homolog
(PTEN), the major negative regulator of the PI3K/Akt
pathway, is a tumor suppressor gene that is localized on
chromosome 10q23. PTEN is dual specific phosphatase
that is involved in controlling cell cycle and apoptosis
[30]. The PI3K signaling pathway is frequently aberrantly
activated in tumors by mutation or loss of the 3'phosphol-
ipid phosphatase. Activation of the PI3K/Akt pathway is
associated with three major radioresistance mechanisms:
intrinsic radioresistance; tumor-cell proliferation; and
hypoxia [31].

Forty-two percent of prostatic tumors have abnormal
PTEN/Akt expression [32]. The human prostate cancer cell
lines, PC-3, DU-145 and LNCaP are known to be resistant
to radiation and thus are widely used in clonogenic radia-
tion assays [33]. One of the cell lines, PC-3, was stably


Mechanism


Bcl-2
P53

pAkt
COX-2

Minor
Heat shock proteins 27, 90 [17-20]

Caspase-I [21]

MDM2 [22]

Clusterin [23,24]
Ras [25]


Mitochondrial membrane protein that blocks the apoptotic death of cells
Protein which responds to diverse cellular stresses, regulating cell cycle arrest, apoptosis, senescence,
DNA repair, or changes in metabolism
Protein responsible for cell survival, proliferation, metabolism and angiogenesis
Enzyme responsible for prostaglandin production which is involved in cellular inflammation and
mitogenesis

Family of proteins whose expression is increased when cells are exposed to external stresses (e.g.,
infection, inflammation, hypoxia) and can inhibit apoptosis and activate proteosomes
Protein which is a member of the cysteine-aspartic acid protease (caspase) family and is central to cell
apoptosis as well as inflammation, septic shock, and wound healing
Protein with an autoregulatory negative feedback loop p53 effecting cell cycle, apoptosis and
tumorigenesis
Glycoprotein observed to have both pro- and antiapoptotic functions
Family of proteins responsible for signal transduction and cell to cell communication


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transfected to overexpress Bcl-2 which generated a cell line
even more resistant to radiation than its parental lines
[34]. Of the above cell lines, only DU-145 has wild-type
PTEN and thus suppressed Akt expression (Table 2). Uti-
lizing gene therapy, overexpression of PTEN resulted in
down regulation of Akt. PTEN restoration caused signifi-
cant GI phase arrest in PC-3 cells. Morphologic changes,
most notably apoptotic bodies and reduction in cellular
proliferation, were evident in all cell lines. In addition, a
reduction in the surviving fractions after 2 Gy of radiation
was evident in the majority of cells assayed: PC-3-Bcl-2,
reduction from 60.5% to 3.6%; PC-3-Neo, no reduction;
LNCaP, reduction from 29.6% to 16.3%; and DU-145,
reduction from 32.7% to 25.7%. Thus in select cells, PTEN
restoration sensitized these cells to the killing effects of
radiation [34]. PTEN restoration was tested in a xenograft
model using the human prostate cancer cell line, PC-3-
Bcl-2. PTEN expression inhibited xenograft tumor growth,
however PTEN expression plus radiation (5 Gy) reduced
tumor size to 75% of the untreated tumor (Figure 1).
Combination treatment also enhanced apoptosis, inhib-
ited cellular proliferation, and inhibited tumor-induced
neovascularity [35].

As the next section will illustrate, Bcl-2 expression can
result in the development of radiation-resistant cancers. It
is interesting that Huang and colleagues demonstrated
that PTEN down regulation results in upregulation of Akt
and Bcl-2, thus illustrating the possible cross-talk between
the Bcl-2 and PI3K/Akt pathway [36].


Table 2: Prostate cancer cell lines phenotype

Cell Line p53 status Bcl-2 status


PC-3-Bcl-2
PC-3-Neo
DUI45
LNCaP


Mutant
Mutant
Mutant
Wild type


Overexpression
Expression
Wild type
Overexpression


PTEN status

Deleted
Deleted
Wild type
Deleted


Unlike for the other molecules we will discuss, limited
clinical information is available regarding PI3K/Akt/PTEN
conferring radiation resistance in actual human samples.
Thus, further studies correlating abnormalities in the
PI3K/Akt/PTEN pathway and the development of radia-
tion-resistant disease are needed. If this correlation can be
demonstrated clinically, the PI3K/Akt/PTEN pathway may
prove to be a desirable therapeutic target that could be
used combination with radiation therapy.

Bcl-2
After p53 the next most commonly discussed genetic aber-
ration associated with radiation resistance is overexpres-
sion of Bcl-2. Bcl-2 is the founding member of a protein
family composed of regulators of programmed cell death
in both normal and abnormal cells. Bcl-2 is a pro-survival
multidomain protein that regulates apoptosis by prevent-
ing the release of pro-apoptotic factors from mitochon-
dria (e.g., cytochrome c) and subsequent activation of a
caspase cascade [37-39]. The role of Bcl-2 as an antiapop-
totic molecule is significant in prostate cancer because of
the level of tenacity and resistance it grants to a tumor. As
a result of these characteristics, it is associated with tumor
aggressiveness [40-43].


In vitro, exposure of cancer cells to low, non-toxic doses of
,I radiation resulted in the up regulation of Bcl-2, indicating
these cells were attempting to adapt to the harmful envi-
. ronment. To confirm that Bcl-2 expression is associated
with radiation resistance, human prostate cancer cells PC-
3 were stably transfected to express Bcl-2 and subjected to
0 10 20 30 40 50 0-6 Gy radiation. The Bcl-2 expressing clone grew more
Days colonies at higher doses of radiation than the control
clones (Figure 2). Utilizing these same cell lines, our
adapted Anai, 2006) group down regulated Bcl-2 expression in this model and
studied its effects with and without radiation in vitro and
I in vivo. Solely targeting Bcl-2 produced no cytotoxic effects
of PTEN expression and radiation therapy on and was associated with G1 cell cycle arrest. The combina-
wth of human prostate cancer xenograft tion of knock-down of Bcl-2 with irradiation sensitized
s. In PC-3 Bcl-2 tumors, a modest, but significant,
,n of tumor growth was demonstrated in Ad-PTEN the cells to the killing effects of radiation. In addition,
compared to other treatment groups, the addition of both PC-3-Bcl-2 and PC-3-Neo xenografts in mice treated
on to Ad-PTEN therapy further inhibited tumor with the combination of targeting Bcl-2 and irradiation
mock no XRT, X; mock + XRT, open square; were >3 times smaller by volume than were xenografts in
N no XRT, open triangle; AdPTEN + XRT, open dia- mice treated with either modality alone (Figure 3). The
Adapted from Anai, 2006). combination therapy was also associated with increased


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(A


Figure
Effect
the gr
tumor!
inhibition
alone. C
irradiati
growth.
AdPTEb
mond. (


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Clonogenic Assay for cell lines


100 M -2
_f PG3B*2


Figure 2
Clonogenic assay of a panel of human prostate can-
cer cell lines subjected to irradiation. Utilizing clono-
genic assay, gold standard for monitoring cell survival after
irradiation, it was demonstrated that PC-3 cells that overex-
pressed Bcl-2 were the most radioresistant cells assayed.


rates of apoptosis, decreased rates of angiogenesis, and
decreased rates of proliferation [44].

Resistance to radiation therapy in human prostate cancers
has been strongly linked to Bcl-2 [45-47]. In a study
including 20 men who were radio-naive and underwent
prostatectomy for prostate cancer, no patient in this group
was Bcl-2 positive. Among 20 men in the same study who
were radio-recurrent and underwent prostatectomy for
prostate cancer, 55% of the tumors were Bcl-2 immunop-
ositive (p = 0.0004) [48]. Further evidence as to the
importance of the expression of key proteins in the Bcl-2
family comes from a prospective, randomized, radiation
dose escalation trial that treated 305 men with localized
prostate cancer with either 70 or 78 Gy. Tumors were
stained using a panel of markers from the Bcl-2 family of
proteins including Bcl-2, Bcl-x (antiapoptotic Bcl-2 family
member) and Bax (proapoptotic Bcl-2 family member).
In the cohort, overexpression of Bcl-2 was observed in
16% of patients; altered Bax expression was observed in
23% of patients; and altered Bcl-x expression was
observed in 53% of patients. Kaplan-Meier survival esti-
mates of freedom from biochemical failure (bNED) and
the log-rank tests revealed significantly lower rates in asso-
ciation with positive Bcl-2 and altered Bax staining. No
correlation was observed between Bcl-x staining; and
bNED. Cox proportional hazards multivariate analysis
confirmed that Bcl-2 and Bax were independent of pre-
treatment PSA level, Gleason score and disease stage in
predicting bNED [49]. Recently Khor and associates
reported on the immunohistochemical results for Bcl-2 in
586 high-risk prostate cancer patients prospectively rand-
omized to the Radiation Therapy Oncology Group


(RTOG) trial 92-02 in which XRT was given in conjunc-
tion with a short course (STAD) or long course (LTAD) of
androgen deprivation therapy. Bcl-2 was positive in
45.6% cases, and Bax expression altered in 53.9% cases.
Bcl-2 overexpression was not independently related to
biochemical failure, local failure, distant metastasis,
cause-specific mortality or overall mortality, although the
relative risks (RR) for failure were higher for overall mor-
tality, cause-specific mortality, distant metastasis, and
local failure, 1.24 (0.96-1.60), 1.24 (0.79-1.94), 1.37
(0.90-2.08) and 1.27 (0.78-2.07), respectively. In univar-
iate analyses, altered Bax expression was not significantly
associated with any of the end points tested either,
although there was a trend for treatment failure (RR, 1.30;
95% CI, 0.97-1.73; p = 0.0806). However in the multivar-
iate analyses, abnormal Bax expression was significantly
associated with overall failure (RR, 1.43; 95% CI, 1.05-
1.95; p = 0.0226) and marginally with biochemical failure
(RR, 1.37; 95% CI, 0.96-1.97; p = 0.0851) [50].

Furthermore, the most common practice treatment for
patients with locally advanced prostate is radiation ther-
apy in conjunction with androgen deprivation therapy
(ADT). ADT treatment triggers an overexpression of Bcl-2
which can lead to androgen independence, a condition
associated with advanced prostate cancer [51]. The com-
pelling data presented above illustrates the significance of
the Bcl-2 family in conferring radiation resistance in pros-
tatic tumors. Strategies designed to favorably alter this
family of survival proteins to enhance the antitumor effect
of radiation warrant further clinical evaluation.

p53
The importance ofTP53 (p53) gene as a molecular marker
in cancer is demonstrated by the finding that mutations of
p53 occur in approximately half of all human malignan-
cies [52]. p53 is a well known protein that acts as a "mas-
ter watchman" in cell-cycle arrest, programmed cell death,
and DNA repair [53-55]. Though some researchers have
reported the response to radiation is independent of the
cell's p53 status [56], numerous reports are available that
demonstrated increased cell kill with irradiation in cells
expressing p53 [57].

Gene therapy strategies based on p53 have been shown to
reduce tumorigenicity and promote apoptosis in a variety
of cell lines and xenograft tumors including colon, head
and neck, ovary, and brain [58-66]. Resistance to apopto-
sis due to cell-cycle disruption has been found to be vari-
ably dependent on the existence of p53. p53 is therefore
an important protein in the sensitization of prostate can-
cer to radiation therapy. In an elegant experiment by
researchers at MD Anderson Cancer Center, the effects of
adenoviral-mediated p53 transgene expression on the
radiation response of two human prostate cancer cell


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(A) PC-3-Bcl-2 Tumor growth
?- 1200
E
S1000
E
I 800

600
0
E
400

200

0

(B) PC-3-Neo Tumor growth

1200


1000

800

600

400


200

0


(Adapted Anai, 2007)


Days


0 10 20 30 40 50 60


0 10 20 30 40 50 60 Days


Figure 3
Effect of BcI-2 targeted therapy in combination with radiation therapy on the growth of human prostate can-
cer xenograft tumors. (A) In PC-3 Bcl-2 tumors, growth inhibition was present with antisense Bcl-2 oligonucleotide (ASO)
alone or radiation alone. Combining ASO and radiation significantly inhibited of tumor growth compared to other treatment
groups. (B) In PC-3-Neo tumors, similar results were evident with combinational therapy resulting in an additive effect with
irradiation. mock no XRT, open circle; mock + XRT, X; ASO no XRT, open square; ASO + XRT, open diamond. (Adapted
from Anai, 2007).


lines, LNCaP and PC3 lines, was examined. After correct-
ing for the effect of Ad5-p53 on plating efficiency, the sur-
viving fraction after 2 Gy of radiation was reduced more
than 2.5-fold, from 0.187 to 0.072, with transgene p53
expression in the LNCaP cell line. Surviving fraction after
4 Gy was reduced greater than 4.5-fold, from 0.014 to
0.003, after Ad5-p53 treatment. In the PC3 cell line, Ad5-
p53 reduced surviving fraction after 2 Gy 1.9-fold from
0.708 to 0.367 and 6-fold at the surviving fraction after 4
Gy from 0.335 to 0.056. Lastly in both cell lines, the com-
bination of Ad5-p53 plus radiation (2 Gy) resulted in


supra-additive apoptosis (approximately 20% for LNCaP
and approximately 15% for PC3 at 50 MOI) above that
seen from the controls [67]. Other groups have been able
to corroborate the results of this in vitro experiment in
human prostate cancer cells [68]. In a follow-up to that
study, Cowen presented the in vivo results in PC3 and
LNCaP xenograft model treated with intratumoral p53
injection and 5 Gy radiation. The time for the PC3 tumors
to reach 500 mm3 were calculated as 10.7 days (+/- 0.7)
for the saline control, 15.6 days (+/- 1.6) for Ad5-p53,
14.6 days (+/- 1.5) radiation therapy, and 31.4 days (+/-


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5.3) for Ad5-p53 plus XRT. Thus, Ad5-p53 plus radiation
significantly retarded the growth of xenograft prostate
tumors. Furthermore in LNCaP xenograft tumors where
serum PSA levels were used to judge treatment efficacy,
treatment with Ad5-p53 plus 5 Gy resulted in significantly
fewer PSA failures (<30%), as compared with failures with
Ad5-p53 alone (64-73%) and the other controls (approx-
imately 80-100%) [69]. These studies indicate that pros-
tatic tumor growth could be inhibited with the over
expression of p53 in combination with radiation therapy.

Several small, retrospective studies in men with prostate
cancer have suggested that abnormal p53 expression is
associated with poor outcomes [70-74]. In addition, pro-
spective clinical trials have clearly demonstrated the prog-
nostic implications of abnormally expressed p53 in men
treated with XRT. For example, tissue samples from 777
men enrolled in RTOG 92-02, the seminal study that com-
pared STAD with XRT to LTAD + XRT, with locally-
advanced prostate cancer were analyzed for p53 expres-
sion. Abnormal p53 expression was defined as 20% or
more tumor cells with positive nuclei. Abnormal p53 was
detected in 168 (21.6%) of 777 cases and was signifi-
cantly associated with cause-specific mortality (adjusted
hazard ratio [HR] = 1.89; 95% confidence interval [CI]
1.14 3.14; p = 0.014) and distant metastasis (adjusted
HR= 1.72; 95% CI 1.13-2.62; p = 0.013). When patients
were divided into subgroups according to assigned treat-
ment, only the subgroup of patients who underwent
STAD + RT showed significant correlation between p53
status and cause-specific mortality (adjusted HR = 2.43;
95% CI = 1.32-4.49; p = 0.0044). This increase in cause-
specific mortality could be reduced with LTAD [73]. Sim-
ilarly, in RTOG 86-10, a phase III trial of Zoladex and
flutamide in locally advanced carcinoma of the prostate
treated with definitive radiotherapy, patients were
assessed for the prognostic significance of abnormal p53
expression. One hundred twenty-nine (27%) of the 471
patients entered in the trial had sufficient tumor material
for analysis. Abnormal p53 protein expression was
detected in the tumors of 23 (18%) of these 129 patients.
Statistically significant associations were found on a mul-
tivariate analysis between the presence of abnormal p53
protein expression and increased incidence of distant
metastases, decreased progression-free survival, and
decreased overall survival. No association was found
between abnormal p53 protein expression and the time to
local progression [75].

In addition, D'Amico reported the results of CALGB 9682
in which 180 men with clinical stage Tic-T3cNOM0 aden-
ocarcinoma of the prostate. They were treated with ADT
and assessed with endorectal magnetic resonance imaging
(eMRI) for change in tumor volume (TV) and associated
PSA outcome. Of these subjects, 141 had sufficient tissue


to assess p53 expression. After a median follow-up of 6.9
years and adjusting for PSA level, Gleason score, clinical
stage, and change in tumor volume, men with abnormal
p53 expression compared with normal were at increased
risk of PSA failure (hazard ratio [HR]: 2.8; 95% confi-
dence interval [CI]) [76].

Knowing the significance of p53 expression, the next hur-
dle is whether we can manipulate p53 expression to
obtain more favorably outcomes. Researchers studying
other tumor types have published on the success of this
approach [77]. For prostate cancer, only one significant
trial stands out. In a phase I/II clinical trial, investigators
at MD Anderson Cancer Center, administered intrapros-
tatic injections of Ad-p53 on three separate occasions
prior to radical prostatectomy in subjects with high-risk
localized prostate cancer. There were no grade 3 or 4
adverse events related to p53 administration. Of the 11
patients with negative baseline immunostaining for p53
protein, 10 had positive p53 immunostaining after the
administration of p53, and 8 had an increase in apoptotic
cells [78]. Though adenoviral gene therapy has its limita-
tions, this study is an excellent example of proof of the
concept that p53 expression may restore the cell's normal
function which can then assist in the eradication of the
tumor. Further research is needed to explore targeting p53
in conjunction with radiation therapy in the treatment of
prostate cancer.

COX-2
Cyclooxygenase is a family of isozymes that convert ara-
chidonic acid to prostaglandins and other eicosanoids.
COX-1, ubiquitously expressed in almost all tissue, is
important for the maintenance of homeostatic function
[79]. COX-2 has been well-characterized as a key compo-
nent in the inflammatory pathway of such disorders as
rheumatoid arthritis and Parkinson's Disease [80,81]. In
addition, COX-2 is overexpressed in 80% of cancers of the
breast, colon, esophagus, liver, lung, pancreas, cervix,
head and neck, and prostate [82-85]. COX-2 is induced by
growth factors, tumor promoters, and cytokines and is
subject to transcriptional and translational regulation and
degradation [86]. Thus, COX-2 is another important cell
survival factor. Overexpression of COX-2 or its related
products confers resistance to cells undergoing chemo-
therapy or radiation therapy induced apoptosis.

Similar to what was reported for Bcl-2, cancer cells
exposed to low doses of radiation up-regulated COX-2
expression as a possible means to survive the radiation
exposure [87]. As described above, therapeutic gamma
radiation exerts its effect on the function and/or survival
of cells by interacting with biologically important mole-
cules, either directly or indirectly. Exposure to radiation
can directly affect DNA structure, which can in turn influ-


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ence the subtle balance of the expression of genes whose
products are involved in promoting cell survival or trigger-
ing cell death. Interestingly, other environmental stres-
sors, including hypoxia and acidosis, resulted in an up-
regulation of COX-2 expression [87].

In a series of in vitro clonogenic assays, the human pros-
tate cancer cell line LNCaP was stably transfected to over-
express COX-2. LNCaP-COX-2 cells were significantly
more resistant to radiation therapy compared to LNCaP-
Neo cells. However, the addition of celecoxib, a selective
COX-2 inhibitor that blocks the production of prostaglan-
dins primarily via inhibition of cyclooxygenase-2, sensi-
tized LNCaP-COX-2 cells to the cytocidal effects of
radiation. Moreover, we confirmed that COX-2 overex-
pression was associated with overexpression of pAkt and
CA IX, two key genes associated with poor tumor response
to radiation [87]. Other preclinical reports have con-
firmed that COX-2 overexpression and/or subsequently
targeting COX-2 with these COX-2 inhibitors may render
cells susceptible to the killing effects of the radiation [88-
911.

Although limited in vivo data are available as to the signif-
icance of COX-2 overexpression as related to its ability to
confer radiation resistance, recently Khor and colleagues
reported the immunohistochemical results for COX-2 in
586 patients enrolled in Radiation Therapy Oncology
Group (RTOG) 92-02. As previously stated, in the RTOG
92-02 trial patients were randomly assigned to treatment
with STAD plus radiotherapy or LTAD plus radiotherapy.
In multivariate analyses, the intensity of COX-2 staining
as a continuous covariate was an independent predictor of
distant metastasis (hazard ratio [HR] 1.181; 95% CI
1.077-1.295); biochemical failure (HR 1.073 [1.014-
1.134]); and any failure (HR 1.068 [1.015-1.124]) [92].

Both the Bcl-2 pathway and COX-2 pathway overlap at
phosphatidylinositol 3-kinase (PI3K)/Akt, making these
molecules attractive targets. The above data support a piv-
otal role for COX-2 expression in tumors treated with irra-
diation and thus justify studies to evaluate the clinical
application of targeting COX-2 in patients undergoing
radiation therapy as a treatment for prostate cancer.

Future Directions
The expression of the aforementioned genes has been reli-
ably linked to the development of radiation-resistance
cancers. It is feasible to conclude that a litany of other sur-
vival genes may be associated with the development of
radiation resistant cancers. Limited data are available
from the use of high throughput technology (e.g.,
genomic and proteomic) in the discovery of new biomar-
kers of which influence resistance to radiation in prostate
tumors. As is clearly evident here, no single molecule is


responsible for the development and growth of radiation-
resistance prostate cancer. As is common in the develop-
ment of primary tumors, multiple genes are aberrant. The
importance of the redundancy in the aberrant genes is that
they are likely to ensure the survival of tumors. The ability
to target more than one gene is an attractive approach. The
recent flourish in the clinical development of more than
30 targeted protein kinase inhibitors designed to inhibit
angiogenesis, tumor growth and progression attests to this
[93,94]. Specifically, Imatinib mesylate [95], Sorafenib
[96], Sunitinib malate [97], and Temsirolimus [98] have
demonstrated efficiency in solid tumors including renal
cell carcinoma. Due to the multiple targets inhibited by
these multikinase inhibitors, effects in different tumor
types are likely to be mediated through a variety of mech-
anisms. Limited forays combining these inhibitors with
radiation therapy in a variety of tumor types have pro-
duced encouraging results [99]. We clearly recognize the
roadblocks in designing clinical trials to test new agents
for radiosensitizing since it is difficult to obtain tissue
after radiation therapy for prostate cancer. However, the
question begs consideration, can targeted therapy effec-
tively treat these cancers? Similar to what we have seen in
other cancers (e.g., testis, lymphoma, colon), optimal
therapy will consist of a therapeutic 'cocktail' involving
traditional chemotherapy and the new targeted therapies
in conjunction with radiation therapy, which has also
evolved over the past decade.

The importance of incorporating these targeted therapies
may be similar to incorporating ADT with radiation ther-
apy. Preclinical studies have demonstrated that ADT can
induce apoptosis and inhibit angiogenesis [100]; out-
comes that may be potentitated by radiation therapy.
These preclinical studies subsequently were translated
into a clinical therapeutic advantage in men with high risk
localized prostate cancer. Only four targeted therapeutic
trials are currently open (Table 3) in men with high risk
prostate cancer treated with radiation therapy. A more
concerted attempt must be made to bring promising tar-
geted therapeutics to clinical trial to determine treatment
efficacy.


Table 3: Accruing Clinical Trials Combining Targeted Therapy
with radiation Therapy


Agent

Sunitinib
SU5416
Bevacizumab

RADOOI


Institute

MD Anderson Cancer Center
University of Chicago
1) Benaroya Research Institute
2) Virginia Mason
Shelba Medical Center


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Conclusion
Radiation therapy continues to be one of the more popu-
lar treatment options for localized prostate cancer. How-
ever, recent studies suggest that abnormal expression of
Bcl-2, p53, Akt/PTEN and cyclooxygenase-2 may render
tumors resistant to the killing effects of radiation. How-
ever modulation of these molecules by a variety of mech-
anisms (e.g., gene therapy, antisense oligonucleotides,
small interfering RNA) may enhance the efficacy of radia-
tion therapy by modifying the activity of these key cell
proliferation and survival pathways.

List of abbreviations
MOI: Multiplicity Of Infection; Ad: adenovirus; PTEN:
Phosphatase and tension; PI3K: Phosphatidylinositol 3
kinase; MAP kinase: mitogen-activated protein kinases;
PKA, PKB, PKC: protein kinase A, B and C; RTOG: Radia-
tion Therapy Oncology Group; STAD: short course of
androgen deprivation therapy; LTAD: long course of
androgen deprivation therapy; eMRI: endorectal magnetic
resonance imaging (eMRI); TV: tumor volume; Gy: Gray;
PSA: prostate specific antigen; XRT: radiation therapy;
bNED: biochemical no evidence of disease; ADT: andro-
gen deprivation therapy; HR: hazard rates; CI: confidence
interval.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
MG collected data and drafted the manuscript. SP assisted
in collecting data and revising the manuscript. CJR con-
ceived the project, and participated in its design and coor-
dination. All authors read and approved the final
manuscript.

Acknowledgements
Joanne Clarke for her review and editing of the manuscript.

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