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Gene Ther Mol Biol Vol 7, 113-133, 2003
Current progress
in adenovirus mediated gene therapy for patients with prostate carcinoma
Review Article
Ahter D.
Sanlioglu1,3, Turker Koksal2,3, Mehmet Baykara2,3, Guven Luleci1,3, Bahri
Karacay4 and Salih Sanlioglu1,3,*
1Departments of
Medical Biology and Genetics, 2Department of Urology and 3The Human Gene
Therapy Unit of Akdeniz University, Faculty of Medicine, Antalya, Turkey,
07070; 4Department of Pediatrics, University of Iowa, College of Medicine, Iowa
City, IA, 52240, USA
__________________________________________________________________________________
*Correspondence:
Salih Sanlioglu V.M.D., Ph.D., Director of The Human Gene Therapy Unit of
Akdeniz University, Faculty of Medicine, B- Block, 1st floor, Campus, Antalya,
07070 Turkey; Phone: (90) 242-227-4343/ext: 44359, Fax: (90) 242-227-4482;
e-mail: HYPERLINK "mailto:sanlioglu@akdeniz.edu.tr" sanlioglu@akdeniz.edu.tr
Key words:
Prostate cancer, adenovirus, gene therapy, immunomodulation, apoptosis, inducible
promoters
Received: 1 July
2003; Accepted: 11 July 2003; electronically published: July 2003
Summary
Prostate cancer
is the most frequently diagnosed male cancer in the world. Like all cancers,
prostate cancer is a disease of uncontrolled cell growth. In some cases tumors
are slow growing and remain local, but in others they may spread rapidly to the
lymph nodes, other organs and especially bone. Although surgery and radiation
can cure early stages of organ confined prostate carcinoma (stages I and II),
there is no curative therapy at this time for locally advanced or metastatic
disease (stages III and IV). The likelihood of postsurgical local recurrence
increases with capsular penetration as detected in 30 % of the patients at the
time of radical prostatectomy. Moreover, 10-15 % of patients have metastatic
cancer at the time of diagnosis. Considering the fact that 60 % local
recurrence is observed in patients receiving radiation therapy with or without
adjuvant hormonal ablation therapy, it is generally believed that androgen
ablation therapy simply delays the progression of prostate carcinoma to a more
advanced stage. In addition, the overall ten-year survival rate of patients
with locally recurrent prostate cancer is only around 35 %; thus; the ultimate
progression into androgen independent prostate carcinoma appears to be
inevitable. Gene therapy arose as a novel treatment modality with the potential
to decrease the morbidity associated with conventional therapies. Therefore,
gene therapy is expected to lower the incidence of tumor recurrence and finally
improve the outcome of patients with recurrent and androgen independent
prostate carcinoma. Viral vectors are most commonly used for the purpose of
gene therapy. Currently, there are a total of 40 clinical trials being
conducted using viral vectors for the treatment of prostate carcinoma. 22 out
of 40 clinical protocols (55 %) approved for the treatment of prostate cancer
utilize adenovirus vectors. Most of these adenovirus mediated therapeutic approaches
employ either selectively replicating adenoviruses or suicide gene therapy
approaches. In this review, we mainly concentrated on the progress in
adenovirus mediated gene therapy approaches for prostate cancer. Analysis of
the death ligand mediated gene therapy approach was also discussed in detail,
while our novel findings were incorporated as an example for up-to-date
approaches used for adenovirus mediated gene therapy against prostate
carcinoma.
I. Introduction
Prostate cancer
is the second leading cause of death in men from cancer following lung
carcinoma with an annual mortality rate of 38,000 (Yeung and Chung, 2002).
There are 200,000 newly diagnosed cases of prostate carcinoma every year in the
United States alone (Boring et al, 1994; Greenlee et al, 2001). As a result,
prostate carcinoma is claimed to be the most frequently diagnosed male cancer
in the United States (Powell et al, 2002). Despite the fact that there has been
a considerable effort for screening and early detection of prostate cancer in
recent years, the lifetime risk of being diagnosed with prostate cancer is
still reported to be 1 in 5 (Grumet and Bruner, 2000). Several hundred clinical
studies using experimental or approved chemotherapeutics failed to improve
survival rates of patients with prostate cancer (Devi, 2002). Because prostate
cancer is a heterogeneous disease, treating patients with prostate cancer still
remains a formidable task. In addition, the molecular mechanism responsible for
the onset of the disease is poorly understood. However, earlier detection of
prostate cancer has been associated with an improved outcome (Perrotti et al,
1998). Thus, the detection of prostate cancer at an earlier stage remains to be
the most realistic chance for therapy.
For this purpose,
different molecular screening methods (Ross et al, 2002a, 2002b) have been
employed, but the most effective method is yet to be established. The most
commonly used screening assays are based on the detection of up-regulated
prostate specific markers such as prostate specific antigen (PSA). Currently,
prostate specific antigen, (Farkas et al, 1998) when it is used in conjunction
with other markers such as Gleason Scoring (Koksal et al, 2000) and TNM grading
(Schroder et al, 1992), is considered to be a valuable tool to evaluate the
histological grade of prostate carcinomas (Xess et al, 2001). As a result,
patients were provided with various treatment options based on the results
obtained with these parameters. These treatment options included but were not limited
to operation, (Klotz, 2000b) radiotherapy, (Do et al, 2002) chemotherapy (Wang
and Waxman, 2001) and hormone therapy (Klotz, 2000a; Smith et al, 2002).
Regrettably, these conventional treatment modalities could not decrease the
casualties from prostate cancer (Hsieh and Chung, 2001). Hence, there is a
great need for development of novel treatment modalities to fight against
prostate cancer. These remorseful facts ignited the initiation of gene therapy
trials for prostate carcinoma (Sanda, 1997). So far, various viral vectors
including lentivirus (Yu et al, 2001a), herpes simplex virus (Jorgensen et al,
2001), adeno-associated virus (Vieweg et al, 1995) and adenovirus (Loimas et
al, 2001) were tested as carriers for therapeutic genes against prostate cancer.
Other types of viruses such as Semliki Forest virus and Sindbis virus were also
tested for gene delivery to prostate cancer cells (Loimas et al, 2001), but
these viruses were unable to transduce prostate cells efficiently. Due to its
antigenic properties and tissue transduction characteristics, adenovirus arose
as a favored transporter vector. The exploitation of the tissue specific
promoter in gene therapy especially eased adenovirus use in clinical trials (Lu
and Steiner, 2000). In this review, we mainly highlighted the progress in
adenovirus mediated prostate cancer gene therapy within the last three years
with a particular emphasis in death ligand mediated gene therapy approach.
II.
Immunomodulation
Tumors exhibit
some degree of immunogenicity and the human immune system responds to these
tumor specific antigens by mounting humoral and cellular responses, which are
essential for the eradication of tumors. Adenovirus is commonly used for the
delivery of genes encoding tumor-associated antigens in order to augment
tumor-specific immune responses. However, antiviral immunity against adenovirus
is a big concern, challenging its application in gene therapy. Various methods
were employed in order to get around the antiviral immunity barrier to increase
the efficacy of adenovirus mediated gene delivery. One of these methods
involves the testing of a collagen-based matrix (Gelfoam) (Siemens et al,
2001). Coinjection of Gelfoam with adenovirus vectors carrying
prostate-specific antigen (Ad5-PSA) into mice naive to PSA but immune to
adenovirus, relinquished the inhibitory effects of adenoviral immunity on CTL
activation. Viral vectors are also being tested to deliver tumor specific
peptides into dendritic cells (DCs) to evoke an immune response. The degree of
immune response generated relies on the functionality of DCs following viral
transduction. To prove this, adenovirus and retrovirus vectors were compared on
the basis of their influence on the functionality of DCs (Lundqvist et al,
2002a). Adenovirus-transduced monocyte-derived DCs (MO-DCs) stimulated
allogenic lymphocytes and produced high levels of TNF and IL12. In addition,
the expression of NF-(B and antiapoptotic molecules such as Bcl-X(L) and Bcl-2
(Lundqvist et al, 2002b) were also increased in adenovirus-transduced MO-DCs.
Consequently, these cells became more resistant to spontaneous as well as
Fas-mediated cell death. In contrast, retroviruses failed even to transduce
MO-DCs. Although CD34(+) cell-derived DCs were transducable with retroviruses
to a lesser extent, they were less potent in their ability to stimulate
allogenic lymphocytes in comparison to nontransduced DCs. These results suggest
that adenovirus transduction of DCs increased the survival and the potency of
DC mediated activation of the immune system. This might be important for
prolonging the antigen presentation to generate a greater degree of immune
response.
Cytokine
stimulated tumor infiltrating macrophages also play a major role in the
generation of the cellular immune response against the tumor. The role of
tumor-infiltrating macrophages in IFN-(-induced host defense against prostate
cancer was revealed using xenograft mice models injected with adenovirus
carrying IFN-( gene (Zhang et al, 2002a). Injection of an adenoviral vector encoding
murine IFN-( (AdIFN-() directly into the tumor suppressed the growth of PC-3MM2
tumors as well as prevented metastasis and prolonged the survival of
tumor-bearing mice. Based on immunohistochemical staining, AdIFN-( infection
resulted in the reduction of microvessel density of the tumor and increased
apoptotic cell death (Cao et al, 2001). On the contrary, macrophage-selective
anti-Mac-1 and anti-Mac-2 antibodies significantly reduced the antitumor effect
of AdIFN-( induced therapy. Therefore, it was concluded that tumor-infiltrating
macrophages must be involved in IFN-( induced suppression of tumor growth and
metastasis.
III. Suicide Gene
Therapy
Suicide strategy
is a combined treatment modality involving chemotherapy and the gene transfer
technology. The underlying principle is to limit the cytotoxicity of a drug to
the local area of the tumor. To achieve this, the cDNA of a prodrug-converting
enzyme is delivered into the tumor using viral vectors followed by regional or
systemic application of the corresponding prodrug. As soon as the prodrug
reaches the tumor, it is taken up and converted to a cytotoxic drug by tumor
cells expressing the prodrug-converting enzyme. For example, 5-Fluorouracil
(5-FU) is widely used as a chemotherapeutic agent for the treatment of various
malignancies. Although clinical trials have been conducted, so far 5-FU
manifested a poor therapeutic index, which drastically limited its clinical use
for cancer therapy. It is still not known whether the lack of success was due
to problems associated with drug delivery or inherent insensitivity of cancer
cells to this metabolite. However, adenovirus (Ad) vector-mediated cytosine
deaminase (CD)/5-fluorocytosine (5-FC) gene therapy had the potential to
overcome pharmacokinetic issues associated with systemic 5-FU administration.
Escherichia coli cytosine deaminase converts the prodrug 5-FC to the cytotoxic
product 5-FU. Adenovirus encoding cytosine deaminase (AdCD) gene was injected
into the prostate cancer cells transplanted orthotopically on mice followed by
the systemic use of 5-FC in order to investigate the antitumor and
anti-metastatic effects of this approach (Zhang et al, 2002c). An effective
inhibition on tumor growth and metastasis was observed through in situ
injection of AdCD followed by systemic use of 5-FC in the xenograft mouse model
of prostate cancer. The use of E. coli uracil phosphoribosyltransferase (UPRT),
a pyrimidine salvage enzyme, which modifies 5-FU into 5-fluorouridine
monophosphate, improved the activity of AdCD through enhancing the anti-tumoral
effect of 5-FU. In order to assess the efficacy of the combined suicide gene
therapy approach, two separate adenovirus constructs expressing either the E.
coli CD or E. coli UPRT genes were infected into androgen refractory prostate
cancer cell line DU145 bearing mice. This combined gene therapy approach
drastically regressed the growth of tumors in these animals better than what
was achieved with AdCD alone (Miyagi et al, 2003).
The most commonly
used prodrug-converting enzyme for clinical approaches is the herpes simplex
virus thymidine kinase gene (HSV-tk). The enzyme thymidine kinase
phosphorylates the prodrug ganciclovir (GCV) to ganciclovir monophosphate,
which is then further phosphorylated by cellular enzymes to ganciclovir
triphosphate, a toxic metabolite and inhibitor of DNA polymerase. The efficacy
of this approach was evaluated in an extended phase I/II study involving 36
prostate cancer patients with local recurrence after radiotherapy. These
patients received single or repeated cycles of replication-deficient adenoviral
mediated HSV-tk plus GCV in situ gene therapy (Miles et al, 2001). The study
concluded that the repeated cycles of in situ HSV-tk plus GCV gene therapy can
safely be administered to patients with prostate cancer who failed radiotherapy
and have a localized recurrence. The therapeutic parameters such as PSA
doubling time (PSADT), the mean PSA reduction (PSAR), and return to initial PSA
(TR-PSA) values were all increased as a response to the treatment, indicating a
therapeutic effect. A combined gene therapy approach using a recombinant
adenovirus containing a fusion gene of CD and HSV-tk controlled by a
cytomegalovirus (CMV) enhancer-promoter was designed to explore new frontiers
in prostate cancer gene therapy (Lee et al, 2002b). Both of the prostate
carcinoma cell lines tested (DU-145 or PC-3 cells) were effectively transduced
and killed by this replication-incompetent adenovirus encoding CD-TK fusion
protein in the presence of prodrugs. The effect of radiation and heat treatment
was also tested using this vector system. Interestingly, heat treatment not
only increased the expression of CD-TK but sensitized prostate cancer cells to
radiation as well. These results suggested that combining heat treatment with
radiation therapy improved the efficacy of the adenovirus mediated suicide gene
therapy approach for prostate carcinoma. The CD-TK fusion fragment was also
cloned into a lytic, replication-competent adenovirus (Ad5-CD/TKrep) and
administered into patients with prostate carcinoma in a Phase I trial. This was
the first gene therapy study in which a replication-competent virus was used to
deliver a therapeutic gene to humans (Freytag et al, 2002a). This study
demonstrated that intraprostatic administration of the replication-competent
Ad5-CD/TKrep virus followed by 2 weeks of 5-fluorocytosine and ganciclovir
prodrug therapy led to the destruction of tumor cells in patients without
safety concerns. In addition, the efficacy and the toxicity of replication-competent
adenovirus-mediated double suicide gene therapy (AdCD-TK) combined with an
external beam radiation therapy (EBRT) approach was tested as a trimodal
treatment modality in a preclinical study (Freytag et al, 2002b). Animals
bearing prostate tumors were first injected with the lytic,
replication-competent Ad5-CD/TKrep virus, then received 1 week of
5-fluorocytosine + ganciclovir (GCV) prodrug therapy supplemented with EBRT.
The results from this study suggested that replication-competent adenovirus-mediated
double suicide gene therapy combined with EBRT is very effective in eliminating
tumors and reducing metastasis in an orthotropic mouse model of prostate
carcinoma.
The efficacy of
another gene-directed enzyme pro-drug therapy based on the Escherichia coli
enzyme purine nucleoside phosphorylase (PNP) was tested in androgen-independent
prostate cancer cells. PNP modifies the prodrug fludarabine to 2-fluoroadenine
(Voeks et al, 2002). In this study, a recombinant ovine adenovirus vector
(OAdV220) with a different receptor choice than that of human adenovirus type 5
carrying the PNP gene under the control of RSV promoter was used for functional
studies. OAdV220 manifested a higher transgene expression compared to human Ad5
vector in infected murine RM1 prostate cancer cells during in vitro studies.
Furthermore, the OAdV220 construct dramatically inhibited subcutaneous tumor
growth when fludarabine phosphate was administered systemically in
immunocompetent mice. Similar results were obtained using human PC3 xenografts
in mice. PNP is also known to convert the prodrug 6MPDR to a toxic purine (6MP)
causing cell death. In order to assess the efficacy of this approach for
prostate cancer, replication-deficient human type-5 adenovirus (Ad5) carrying
the PNP gene (Ad5-SVPb-PNP) was directly injected into PC3 tumors
(Martiniello-Wilks et al, 2002). The specificity and the level of transgene
expression from this recombinant adenoviral vector were controlled by the
promoter from the androgen-dependent, prostate-specific rat probasin (Pb) gene
hooked up to the SV40 enhancer (SVPb). Unexpectedly, the SVPb element confirmed
substantial prostate specificity even in the absence of androgens. Intratumoral
delivery of Ad5-SVPb-PNP followed by 6MPDR administration significantly
suppressed the growth of human prostate tumors in nude mice. These results
suggested that Ad5-SVPb-PNP has therapeutic potential even in the absence of
androgens for the treatment of prostate carcinoma.
Another non-toxic
prodrug, CB1954, which is converted to a toxic metabolite by the Escherichia
coli nitroreductase gene (NTR), was tested as a suicide gene therapy approach
for prostate cancer. Adenovirus vector expressing NTR (CTL102) was injected
into subcutaneous prostate cancer xenografts followed by systemic CB1954
administration (Djeha et al, 2001). A clear anti-tumor effect of the approach
was observed. In addition to all the methods mentioned above, a novel approach
inspired from radioiodine therapy for thyroid cancer was developed using sodium
iodide symporter (NIS). NIS is normally exclusively expressed in thyroid
glands. Adenovirus carrying the NIS gene (AdCMVNIS) was constructed and tested
for the treatment of prostate cancer following 131I administration (Spitzweg et
al, 2001). Injection of AdCMVNIS construct to prostate cancer xenografts
manifested highly active radioiodine uptake resulting in a drastic reduction in
the tumor size following 131I administration in nude mice. This new approach
represented an effective and potentially curative modality leading to the
accumulation of therapeutically effective radioiodine in prostate.
Diphtheria toxin
(DT) is known to be a potent inhibitor of protein synthesis. The fact that a
single molecule of DT can result in cell death complicated the utilization of
DT as a suicide gene for cancer therapy. Thus, the feasibility of using DT gene
therapy would greatly be influenced by tissue specific gene expression.
Adenovirus vector carrying the catalytic domain (A chain) of DT under the
control of the prostate-specific antigen (PSA) promoter (Ad5PSE-DT-A) induced
apoptosis in PSA-positive prostate cancer cells in the presence of exogenous
androgen (R1881) (Li et al, 2002a). In addition, Ad5PSE-DT-A injection
regressed the growth of a PSA-positive LNCaP xenograft in nu/nu mice.
Non-PSA-secreting DU-145 cells did not manifest the same effect due to the lack
of activation of PSA promoter in these cells. Therefore, the Ad5PSE-DT-A viral
gene therapy approach might be a viable alternative in the treatment of PSA-secreting
androgen-dependent prostate carcinoma.
IV. Joint
approaches involving immunomodulation-hormonal or radiation therapy in
combination with suicide gene approach
AdHSV-tk suicide
gene therapy was coupled to adenovirus-mediated IL-12 delivery as a combined
gene therapy approach in order to enhance NK activity induced by HSV-tk gene
expression and ganciclovir (GCV) treatment (Hall et al, 2002). This dual
treatment generated radical local and systemic growth suppression in a
metastatic model of mouse prostate cancer (RM-1). The unification of
AdHSV-tk/GCV + Ad.mIL-12 gene therapy approaches resulted in the induction of
apoptosis due to increased expression of Fas and FasL and improved
anti-metastatic activity secondary to a strong NK effect. Intra-tumoral
injection of AdHSV-tk vector followed by systemic ganciclovir or local
radiation therapy or the combination of gene and radiation therapy was
administered to subcutaneously transplanted mouse prostate tumors (Chhikara et
al, 2001). The combined treatment reduced tumor growth by 61% compared to 38%
obtained by single therapy modalities. Combined therapy also increased the mean
survival time. In order to analyze systemic anti-tumor activity, lung
metastases were generated by tail vein injection of RM-1 prostate cancer cells.
While radiotherapy alone had no effect on the metastatic growth, the number of
lung nodules was reduced by 37% following treatment with AdHSV-tk. The
combinational therapy led to an additional 50% reduction in lung colonization.
This was the first study demonstrating a significant systemic effect of
AdHSV-tk administration combined with radiation. A Phase I/II study of
radiotherapy and in situ gene therapy (adenovirus/herpes simplex virus
thymidine kinase gene/valacyclovir) in combination with or without hormonal
therapy in the treatment of prostate cancer was conducted recently (Teh et al,
2001). Based on the preliminary results, no serious side effect of the combined
therapy was observed. This was reported as the first trial of its kind in the
field of prostate cancer, and is expected to enlarge the curative index of
radiotherapy by merging in situ gene therapy.
V. Molecular
signaling pathways modulating the efficacy of adenovirus mediated therapeutic
gene delivery
Expression of
certain hormone and growth factor receptors as well as cytokines and related
downstream molecules can affect the efficacy of adenovirus-mediated gene
therapy for prostate cancer. For example, gonadotrophin-releasing hormone
(GnRH) restrains cell growth of reproductive tissue via gonadotrophin-releasing
hormone receptors (GnRH-Rs) expressed in most cancers of reproductive tissues
like that of prostate. Unfortunately, endogenous GnRH-R expression was not
detected in PC3 cells, indicating that the cells are insensitive to GnRH.
Exogenous expression of high affinity GnRH-R using adenovirus vectors
(AdGnRH-R) facilitated antiproliferative effects of GnRH agonists in prostate
cancer cells (Franklin et al, 2003). In addition, most of the prostate cancer
cell lines overexpress fibroblast growth factors (FGFs). FGF signaling controls
cell proliferation and inhibits cell death. A recombinant adenovirus expressing
a dominant-negative FGF receptor (AdDNFGFR-1) was created in order to determine
the biological significance of altered FGF signaling in human prostate cancer
(Ozen et al, 2001). AdDNFGFR-1 infection of LNCaP and DU145 prostate cancer
cells induced extensive cell death within 48 hours. Some of the prostate cancer
cell lines are androgen dependent (LNCaP) whereas some are androgen independent
(DU145 or PC3). Androgen ablation therapy, surgery, and radiation therapy are
relatively effective in treating androgen dependent prostate carcinoma. However
these treatments were ineffective for androgen-insensitive prostate carcinoma.
Upregulation of IL6 cytokine induced by the constitutive NF-(B and Jun D
activation is one of the distinctive parameters of androgen independent cell
lines (Giri et al, 2001). IL6 is known to function as a proliferation and
differentiation factor for prostate carcinoma. The infection with adenovirus
vectors encoding either the dominant negative form of I(B( gene or Jun D
reduced IL6 gene expression, leading to growth suppression of prostate cancer
cells (Zerbini et al, 2003). Some but not all prostate cancer cells respond to
vitamin D treatment. 1(, 25-Dihydroxyvitamin D(3) (1(, 25-(OH)(2)D(3)) is known
to have significant antiproliferative effects on certain prostatic carcinoma
(PC) cell lines. 1(, 25-(OH)(2)D(3) inhibited cell growth and upregulated p21
expression in PC cell lines such as ALVA-31 and LNCaP (Moffatt et al, 2001).
Stable transfection with a p21 antisense construct abolished the growth
inhibition of ALVA-31 cells without altering vitamin D receptor expression. On
the contrary, adenovirus-mediated expression of a sense p21 cDNA significantly
reduced the proliferation of 1(, 25-(OH)(2)D(3) unresponsive TSU-Pr1 and JCA-1
prostate cancer cell lines. Therefore, Adp21 gene therapy may be useful even
for prostate cancer patients not responding to vitamin D treatment.
Molecular
signaling pathways are also altered in cancer cells. For instance, highly
metastatic tumor cell lines display increased activity for focal adhesion
kinase (FAK). The role of FAK in regulating migration of prostate carcinoma cell
lines with increasing metastatic potential was studied in detail (Slack et al,
2001). Highly tumorigenic PC3 and DU145 cells displayed intrinsic migratory
capacity correlating with an increased FAK expression and activity. On the
contrary, poorly tumorigenic LNCaP cells required a stimulus to migrate.
Inhibiting the FAK/Src signal transduction pathway by overexpressing FRNK
(Focal adhesion kinase-Related Non-Kinase), an inhibitor of FAK activation,
significantly inhibited migration of prostate carcinoma cells. Modulation of
phosphatidylinositol 3'-kinase (PI3'-kinase), leading to Akt activation,
frequently occurs in prostate cancer and disrupts apoptotic signaling induced
by various cytokines such as tumor necrosis factor TNF and TNF-related
apoptosis-inducing ligand (TRAIL). Two prostate cancer cell lines with
constitutively activated PI3'-kinase cascades (LNCaP and PC-3) were examined in
order to study the role of PI3' phosphorylation in cellular response to TNF or
TRAIL alone. Both TNF and TRAIL failed to activate apoptosis in either LNCaP or
PC-3 cells. Interestingly, downregulation of PI3'-kinase/Akt signaling
significantly enhanced the apoptotic activity of both TNF and TRAIL in LNCaP
cells but not in PC-3 cells. Infection with adenovirus delivered PTEN/MMAC1
(phosphatase and tensin homologue/mutated in multiple advanced cancers) reduced
Akt activation, activated apoptosis and sensitized cells to TNF but not to
TRAIL in LNCaP cell line (Beresford et al, 2001). Therefore, it was concluded
that although PI3'-kinase signaling inhibits both TNF and TRAIL mediated
apoptosis, this may only represent one of the several apoptotic resistance
mechanisms in signaling pathways.
Selenium
compounds are known to be potential chemotherapeutic agents for prostate cancer.
NF-(B has been categorized as the key antiapoptotic signaling molecule often
activated in transformed cells. Testing of selenium compounds on DU145 and JCA1
prostate carcinoma cells revealed that these compounds induced apoptosis
through the inhibition of NF-(B pathways in these cell lines (Gasparian et al,
2002b). Increased IKK activity was blamed for constitutive NF-(B activation
responsible for survival of androgen independent prostate carcinoma cell lines
(Gasparian et al, 2002a).
60-80 % of
prostate cancers acquire the PTEN mutation during tumorigenesis. This results
in the constitutive activation of the PI3'-kinase pathway and prostatic cell
proliferation. The loss of PTEN activity is also correlated with the loss of
activity of the FOXO family of forkhead transcription factors such as FKHRL1
and FKHR. Interestingly, these transcription factors are shown to control the
expression of apoptosis inducing ligand TRAIL. Not surprisingly, the expression
of TRAIL was also reduced in PTEN-lacking prostate cancer cells, leading to
decreased apoptosis. Restoration of TRAIL expression using adenovirus-mediated
overexpression of these transcription factors in LAPC4 prostate cancer cell
line induced apoptosis (Modur et al, 2002).
VI. Apoptosis
Modulators
A. The exploitation
of death ligands to induce apoptosis in cancer cells
Apoptosis, known
as programmed cell death (Reed, 2000) is defined as cells preferred form of
death under hectic conditions (Sears and Nevins, 2002). In reality, it is also
a key mechanism for homeostasis throughout embryonic and adult life. Genetic
aberrations disrupting programmed cell death underpin tumorigenesis and drug
resistance. Therefore, the specific activation of apoptosis within tumor cells
could be a highly effective therapeutic intervention for prostate cancer.
Currently, chemotherapy (Stein et al, 2002) and radiotherapy (Wang et al, 2002)
are among the most commonly used treatment modalities against prostate cancer.
The tumor suppressor gene, p53, is required in order for both of these
treatment methods to work as anti-tumor agents (Levine, 1997). However, more
than half of the human tumors acquire p53 mutations during tumorigenesis
(Horowitz, 1999; Zeimet et al, 2000). As a result, tumors lacking p53 display
resistance to both chemotherapy and radiotherapy (Obata et al, 2000).
Intriguingly, death ligands induce apoptosis independent of p53 status of the
cells (Ehlert and Kubbutat, 2001; Norris et al, 2001). Thus, these methods
constitute somewhat of a complementary treatment modality to currently employed
conventional treatments. At present, death ligands are being evaluated as
potential cancer therapeutic agents (Herr and Debatin, 2001). Previously,
several studies using external Fas agonists, anti-Fas antibodies and membrane-bound
FasL failed to induce Fas L mediated apoptosis in prostate cancer cells.
Although the down regulation of c-FLIP expression through the use of anti-sense
oligonucleotides sensitized DU145 cells to an anti-Fas monoclonal antibody
(Hyer et al, 2002), efficient cell killing was not observed by this approach.
However, intracellular expression of FasL using adenoviruses efficiently killed
70-90% of various human prostate cancer cell lines tested (Hyer et al, 2000).
Furthermore, part of this cell killing was attributed to the bystander effect
mediated by FasL carried within the apoptotic bodies and cellular debris (Hyer
et al, 2003). Despite the fact that human prostate cancer cells express
apoptotic FasL, some of the cell lines, such as LNCaP, are resistant to Fas L
mediated cell death. Even so, prior exposure to IFN( sensitized orthotropic
prostate primary tumors to recombinant adenovirus mediated FasL delivery
(Selleck et al, 2003). Despite the fact that tumor necrosis factor (TNF)
(Terlikowski, 2001) and FasL (Nagata, 1997) have been studied extensively and
were shown to effectively induce apoptosis in cancer cells, their systemic use
in cancer gene therapy is not recommended due to the systemic toxicity.
With the
discovery of a novel death ligand, TRAIL/Apo2L, (Wiley et al, 1995; Pitti et
al, 1996) a new era emerged for the deployment of death ligands for cancer gene
therapy (Nagane et al, 2001). The fact that TRAIL does not cause any harm to
normal cells but can selectively induce apoptosis in cancer cells brought up
the possibility of TRAIL testing for systemic use (Griffith and Lynch, 1998).
Five different receptors were identified to interact with TRAIL; TRAIL-R1,
TRAIL-R2, TRAIL-R3, TRAIL-R4 and osteoprotegrin (Abe et al, 2000; Sheikh and
Fornace, 2000). TRAIL-R1 and TRAIL-R2 function as authentic death receptors
inducing apoptosis while TRAIL-R3 and TRAIL-R4 are unable to induce such
signaling but can serve as decoy receptors (Meng et al, 2000). However even
today, no single mechanism has been found to account for TRAIL resistance
observed in normal cells. The soluble form of TRAIL has successfully been
tested and no toxicity due to systemic use was observed in animal models.
However, large quantities of TRAIL were needed in order to suppress the tumor
growth. A replication-deficient adenovirus encoding human TRAIL (TNFSF10;
Ad5-TRAIL) was generated as an alternative to recombinant, soluble TRAIL
protein (Griffith and Broghammer, 2001). Ad5-TRAIL infection into
TRAIL-sensitive prostate tumor cells induced apoptosis through the activation
of Caspase 8 pathways. Normal prostate epithelial cells were not harmed by
Ad5-TRAIL infection. Moreover, in vivo Ad5-TRAIL administration suppressed the
outgrowth of human prostate tumor xenografts in SCID mice. Eight prostate
cancer cell lines (CWR22Rv1, Du145, DuPro, JCA-1, LNCaP, PC-3, PPC-1, and
TsuPr1) and primary cultures of normal prostate epithelial cells (PrEC) were
tested for sensitivity to soluble TRAIL induced cell death in another study
(Voelkel-Johnson et al, 2002). 100 ng/mL of soluble TRAIL administration did
not induce apoptosis in Du145, DuPro, LNCaP, TsuPr1, and PrEC. Interestingly,
treatment with the chemotherapeutic agent doxorubicin sensitized almost all
prostate cancer cells to TRAIL-induced cell death. On the other hand, an
adenoviral vector expressing full-length TRAIL (AdTRAIL-IRES-GFP) killed
prostate cancer cell lines and, unexpectedly, PrEC as well, independent of
doxorubicin cotreatment. This study suggested that the AdTRAIL-IRES-GFP gene
therapy approach, complemented with tissue-specific promoters, would be useful
for the treatment of prostate carcinoma. However, the mechanism of TRAIL
resistance in normal cells is not understood and some prostate cancer cells
appeared to be TRAIL-resistant (Nesterov et al, 2001). In one study, ALVA-31,
PC-3, and DU 145 cell lines were highly sensitive to apoptosis induced by
TRAIL, while TSU-Pr1 and JCA-1 cell lines were moderately sensitive, and the
LNCaP cell line was resistant (Nesterov et al, 2001). Due to the lack of active
lipid phosphatase PTEN, LNCaP cells demonstrated a constitutive Akt activity.
Akt is a negative regulator of the phosphatidylinositol (PI)3-kinase/Akt
pathway. PI3-kinase inhibitors sensitized LNCaP prostate cancer cells to TRAIL.
In addition, adenovirus expressing a constitutively active Akt reversed the
ability of wortmannin to potentiate TRAIL-induced BID cleavage. This suggested
that constitutive Akt activity inhibits TRAIL-mediated apoptosis (Nesterov et
al, 2001).
B. NF-(B
inhibiting approaches used to breakdown TRAIL resistance in prostate cancer
cells
The mechanism of
TRAIL induced apoptosis and resistance is outlined in Figure 1. So far, at
least two different hypotheses that may partly explain TRAIL resistance are
asserted. The first hypothesis advocates that normal cells carry decoy
receptors (TRAIL-R3, TRAIL-R4), which compete with apoptosis inducing TRAIL
receptors (TRAIL-R1, TRAIL-R2) for binding to TRAIL (Pan et al, 1997; Sheridan
et al, 1997). In this hypothesis, it is believed that decoy receptors either
function to dilute out TRAIL ligands (like TRAIL-R3) or supply anti-apoptotic
signals (like TRAIL-R4) to cells. As reported previously, TRAIL-R4 binding
activates the anti-apoptotic NF-(B signaling pathway, leading to the blockade
of TRAIL induced apoptosis (Degli-Esposti et al, 1997). In addition, the
expression of decoy receptors is down-regulated in cancer cells through
promoter hypermethylation leading to differential sensitivity to TRAIL (van
Noesel et al, 2002). However, the link between TRAIL resistance and the
expression of decoy receptors has not been clearly established in human cells
(Griffith and Lynch, 1998). Interestingly, activation of death receptors such
as TRAIL-R1 and TRAIL-R2 also stimulated the NF-(B pathway (Chaudhary et al,
1997; Schneider et al, 1997). Under these circumstances, the reason(s) for
cells undergoing apoptosis despite the induction of anti-apoptotic pathways
through the same death receptors is not fully understood.
Figure 1: A gene
therapy strategy to block anti-apoptotic NF-(B signaling pathway to induce
TRAIL sensitivity in prostate cancer cells. Activation of TRAIL receptor 1 (R1)
or 2 (R2) by trimeric TRAIL ligands leads to the recruitment of Fas associated
death domain protein (FADD) to the membrane. Then, FADD recruits procaspase 8
to form death inducing signaling complex (DISC). DISC induced signaling
activates caspase pathway inducing cells into apoptosis. TRAIL receptor 3 (R1)
and 4 (R4) serve as decoy receptors. R4 activates NF-(B signaling pathways as
well. In addition, NF-(B pathway is also activated by R1 and R2 via
TNFR-associated death domain protein (TRADD) and receptor interacting protein
(RIP). Consequently, NF-(B activation augments expressions of various
anti-apoptotic genes such as cIAP, BclxL and cFlip in addition to R3. c-Flip, a
procaspase 8 homologue, competes with procaspase 8 for binding to FADD. Thereby
it inhibits apoptotic signaling. The expression of adenovirus delivered IKK(KA
mutant prevented the activation of anti-apoptotic NF-(B signaling. This method
sensitized prostate cancer cells to TRAIL.
The second
hypothesis claims the presence of apoptosis inhibitory substances in these
cells. Such a molecule, cFLIP (FLICE Inhibitory Protein), a caspase 8
homologue, has been shown to obstruct death ligand induced apoptosis (Irmler et
al, 1997; Griffith et al, 1998). Intriguingly, NF-(B activating agents
up-regulated cFLIP synthesis (Kreuz et al, 2001). Furthermore, the NF-(B
pathway has been proven to increase TRAIL-R3 synthesis, a decoy receptor for
TRAIL, (Bernard et al, 2001) and the expression of apoptosis inhibitor Bcl-xL
(Hatano and Brenner, 2001; Ravi et al, 2001) resulting in the obstruction of
TRAIL mediated apoptosis. Apoptosis inhibitors such as cIAP are also activated
by NF-(B pathways (Mitsiades et al, 2002). Based on these results, we can
clearly state that the active NF-(B signaling pathway may provide cells with
TRAIL resistance by at least four different ways (Figure 1). Additionally, it
has been reported that a novel tumor suppressor gene, PTEN/MMAC1 (Steck et al,
1997; Simpson and Parsons, 2001) negatively regulated TNF induced NF-(B
activity (Ozes et al, 1999; Mayo et al, 2002) through the IKK complex (Gustin
et al, 2001). The observation in which IKK activity is required for PI3K-Akt
induced NF-(B activation (Burow et al, 2000; Demarchi et al, 2001) confirmed
this report (Madrid et al, 2001; Sizemore et al, 2002). Due to a negative
correlation between the expression of PTEN and the progression of prostate
cancer, advanced prostate cancer cells might have intrinsically higher NF-(B
activity due to the progressive loss of PTEN. Absence of PTEN function may
result in increased Akt activity induced by PI3K. Since NF-(B is a downstream
target for Akt, (Kane et al, 1999; Romashkova and Makarov, 1999; Andjelic et
al, 2000; Jones et al, 2000) TRAIL resistance would ultimately be ensured in
cells by way of the NF-(B pathway. In agreement with this hypothesis, PTEN
sensitized prostate cancer cells to TRAIL induced apoptosis (Yuan and Whang,
2002). Thus, these possible scenarios make NF-(B inhibiting vectors such as
Ad.IKK(KA (Sanlioglu et al, 2001a) or Ad.I(B(SR (Batra et al, 1999; Sanlioglu
and Engelhardt, 1999) ideal candidates for overcoming the TRAIL resistance in
PTEN mutant prostate cancer cells. In a similar manner, TNF induced apoptosis
can also be prevented by NF-(B activation as reported (Beg and Baltimore, 1996;
Van Antwerp et al, 1996). Previously, NF-(B inhibiting approaches such as
adenovirus mediated transfer of IKK( (Ad.IKK(KA) (Sanlioglu et al, 2001a,
2001b) or I(B( (Ad.I(B(SR) (Batra et al, 1999; Sanlioglu and Engelhardt, 1999)
dominant negative mutants were successfully deployed in order to sensitize lung
cancer cells to TNF. Since some tumor cells have intrinsically high NF-(B
activity, which might be responsible for TRAIL resistance, NF-(B blocking
agents can potentially be useful to overcome TRAIL resistance. For example, a
constitutive NF-(B activation was observed in renal carcinoma (Oya et al,
2001). Not surprisingly, melanoma cells having a constitutive NF-(B activity
exhibit TRAIL resistance (Franco et al, 2001). Resistant melanoma cells were
sensitized to TRAIL either with proteasome inhibitors or transfections with
plasmids encoding degradation resistant I(B( protein (Franco et al, 2001). In
accordance with these studies, we have tested if adenovirus mediated NF-(B
inhibiting approach would sensitize prostate cancer cells to TRAIL.
Consequently, adenovirus mediated delivery of IKK(KA mutant (Ad.IKK(KA)
sensitized PTEN mutant prostate cancer cells (PC3) to TRAIL as shown in Figure
2. At first, PC3 cells appeared to be relatively resistant to pro-apoptotic
effects of TRAIL when cells were infected with adenovirus vector encoding
hTRAIL (Ad.hTRAIL) even at an MOI of 1000 DNA particles/cell (Figure 2 Panel
A). Infection with Ad.IKK(KA vector alone did not yield any cell death either
(Figure 2, Panel B). However, when the dose of Ad.hTRAIL vector was kept
constant at an MOI of 1000 DNA particles/cell, increasing the amount of
Ad.IKK(KA construct sensitized PC3 cells to TRAIL mediated apoptosis (Figure 2,
Panel C).
C. Intracellular
proapoptotic regulators
Although caspases
are the effector mediators of apoptosis, the expression of proapoptotic molecules
such as procaspase 3 or 7 using adenovirus constructs did not induce apoptosis
in prostate cancer cells due to the inability of these caspases to undergo
autocatalytic activation (Li et al, 2001). A novel suicide gene therapy
approach was developed using chemically inducible effector caspases to trigger
apoptosis in prostate cancer cells. Cell death was mediated by
replication-deficient adenoviral vector expressing conditional caspase-1
(Ad-G/iCasp1) or caspase-3 (Ad-G/iCasp3) and the caspase activation was
achieved by nontoxic, lipid-permeable, chemical inducers of dimerization (CID)
(Shariat et al, 2001). Aggregation and activation of these recombinant caspases
occurred, leading to rapid apoptosis only after vector transduction followed by
CID administration in both human (LNCaP and PC-3) and murine (TRAMP-C2 and
TRAMP-C2G) prostate cancer cell lines. Subcutaneous TRAMP-C2 tumors displayed
focal but extensive apoptosis following direct injection of Ad-G/iCasp1 in
vivo. In order to express caspase 9 exclusively in prostate, a recombinant
adenovirus carrying iCaspase-9 was constructed with two copies of the androgen
response region (ARR) placed upstream of the probasin promoter elements
(ADV.ARR(2)PB-iCasp9) (Xie et al, 2001b). AP20187 is a chemical dimeric ligand,
which causes dimerization and thereby activation of iCaspase-9 leading to rapid
apoptosis in both dividing and nondividing cells. Testing of
ADV.ARR(2)PB-iCasp9 construct in LNCaP tumor xenografts demonstrated that this
construct induces apoptosis in prostate cancer cells only in the presence of
AP20187.
The proapoptotic members of Bcl- 2 protein
family including Bax, Bak, Bad, and Bik also mediate apoptosis.
Apoptosis-inducing proteins were cloned into adenovirus constructs and shown to
induce apoptosis in prostate cancer cell lines previously.
Figure 2.
Adenovirus mediated IKK(KA expression sensitized PC3 cells to TRAIL mediated
apoptosis. PC3 cells were infected with increasing MOIs of either Ad5hTRAIL
(Panel A) or Ad.IKK(KA (Panel B). In panel C, the dose of Ad.IKK(KA vector was
increased gradually (stated just above each panel) while the amount of
Ad5hTRAIL was kept constant (as indicated under the panel). Cell death was
detected using molecular probes Live and Death Cellular viability and toxicity
kit 48 hours following infection. Numbers indicate viral doses as MOI values of
DNA particles/cell.
However,
overexpression of proapoptotic genes without the use of tissue specific
promoters could result in unwanted apoptosis even in normal cells. In order to
provide tissue specificity, an adenoviral construct was generated containing
Bax cDNA under control of the probasin promoter that included two androgen
response elements (Av-ARR2PB-Bax). Av-ARR2PB-Bax construct drove Bax
overexpression in an androgen-dependent way in androgen receptor (AR)-positive
cell lines of prostatic origin but not in others. The androgen
dihydrotestosterone activated apoptosis in LNCaP cells infected with
Av-ARR2PB-Bax but not in those infected with control vectors. These results
demonstrated that Av-ARR2PB-Bax induced apoptosis was androgen dependent and
limited to AR positive cells of prostatic epithelium. On the other hand, using
a binary co-transfection strategy involving Ad/GT Bax and Ad/PGK-GV16;
overexpression of proapoptotic Bax protein induced apoptosis both in
androgen-insensitive (DU145 and PC3), and androgen-sensitive (LNCaP) cell lines
(Honda et al, 2002). The same binary approach was tested to assess the
consequences of Bcl-2 overexpression in the progression of prostate carcinoma
leading to apoptosis-resistant and androgen-independent phenotype in DU145, PC3
and LNCaP cell lines which represent models of advanced prostate carcinoma. Bax
expression generated by the adenoviral co-transfection system induced apoptosis
even in these Bcl-2 overexpressing cell lines. These results suggest that the
Ad/GT Bax and Ad/PGK-GV16 combined expression system might represent a powerful
gene therapy strategy for the treatment of androgen-independent and
apoptosis-resistant prostate carcinoma. Moreover, monogene and polygene
approaches were compared in an experimental prostate cancer model using
apoptotic genes bad and bax driven by a prostate specific promoter (ARR(2)PB)
in an adenovirus construct (Zhang et al, 2002b). The ARR(2)PB is a
dihydrotestosterone (DHT)-inducible third-generation probasin-derived promoter.
In this study, animals bearing tumors of prostatic origin responded better to
combined bad and bax therapy than either of the vectors alone. Therefore, it
was concluded that polygene therapy involving more than one apoptotic molecule
is more effective in xenograft models of androgen-dependent or independent
prostate cancer than monogene therapy alone. It is also known that
overexpression of anti-apoptotic genes such as Bcl-2 in prostate carcinoma
provides resistance to radiation therapy and androgen ablation. A
second-generation adenoviral vector (ARR2PB.Bax.GFP) was constructed with the
modified prostate-specific probasin promoter (ARR2PB) directing the expression of
a HA-tagged Bax gene in order to restore the balance of Bcl-2 family members to
induce apoptosis in prostate cancer cells (Lowe et al, 2001). ARR2PB.Bax.GFP
vector induced significant levels of apoptosis in LNCaP cells 48 hours
following infection even in the presence of high levels of Bcl-2 protein. No
toxicity in liver, lung, kidney, and spleen was detected by systemic
administration of ARR2PB.Bax.GFP in nude mice. Therefore, a second-generation
adenovirus-mediated, prostate-specific Bax gene therapy appeared to be a very
safe and efficient approach for the treatment of prostate cancer. Another
member of the proapoptotic Bcl-2 family, namely "Bik", was cloned
into adenovirus vectors to explore its therapeutic potential. AdBik infection
also induced apoptosis and suppressed the growth of PC-3 xenografts established
in nude mice (Tong et al, 2001).
Several other genes were also tested for their
ability to induce apoptosis in prostate tumor cell lines as well as in
xenograft models. The antiapoptotic protein CLN3 negatively regulates
endogenous ceramide production, an inducer of apoptotic cell death. CLN3
protein is overexpressed in most of the cancer cell lines tested including
those of prostate (Du145, PC-3, and LNCaP). An adenovirus-expressing antisense CLN3
(Ad-AS-CLN3) blocked CLN3 protein expression in prostate cancer cell lines as
demonstrated by Western Blotting (Rylova et al, 2002). Ad-AS-CLN3 infection
resulted in the inhibition of cell growth and reduction in cell viability of
cancer cells through elevation of endogenous ceramide production. This study
revealed CLN3 as a novel target to induce apoptosis in prostate cancer cells. A
recombinant adenovirus containing pHyde cDNA gene (AdpHyde), a novel gene
cloned from Dunning rat prostate cancer cells, was constructed in order to
study its function (Zhang et al, 2001). Surprisingly, the AdpHyde construct
inhibited the growth of human prostate cancer cells and induced apoptosis
involving the caspase-3 pathway in human prostate cancer tumor xenografts in
nude mice. Ionic movement also influences apoptosis. For instance, K+ efflux is
an early event in apoptosis, which is regulated by K+ channel-associated
protein (KChAP). A recombinant adenovirus encoding KChAP (Ad/KChAP) was
constructed in order to determine if KChAP expression could induce apoptosis in
prostate cancer cells (Wible et al, 2002). The LNCaP cell line displayed a
reduction in cell size upon infection with Ad/KChAP. The Ad/KChAP construct
also induced apoptosis in DU145 cells in a p53 independent manner. In addition,
infection with Ad/KChAP prevented growth of DU145 and LNCaP tumor xenografts in
nude mice.
VII. Tumor
suppressor genes
Aberrations in
the expression of tumor suppressor genes have been one of the key factors
affecting the outcome of cancer therapy. Several studies examined the possible
use of tumor suppressor genes as therapeutic agents for prostate cancer.
Doxorubicin (Dx) is a commonly used chemotherapeutic agent in recurrent
prostate cancer and is a strong inducer of p53 expression leading to
p21(CIP1/WAF1) transactivation. As suggested by previous reports, p21 plays a
role in the modulation of chemotherapy-induced apoptosis, prostate cancer
progression and androgen regulation. Two androgen-regulated human prostate
cancer cell lines (MDA PCa 2b and LNCaP) were exposed to Dx and growth factor
withdrawal in order to investigate if p21 plays a role in the survival of
prostate cancer cells under stress (Martinez et al, 2002). Infection with
adenovirus vectors encoding the antisense strand of p21 reduced p21 levels,
sensitized prostate cancer cells to Dx and facilitated apoptosis in response to
growth factor withdrawal. These results suggest that modulation of p21
pro-survival gene expression via adenovirus constructs sensitizes prostate
cancer cells to chemotherapeutics and androgen withdrawal. Another tumor
suppressor protein, p27, also known as cyclin-dependent kinase inhibitor
(CDKI), is normally expressed in human prostate. However, the majority of human
prostate cancers have reduced levels of p27. The down regulation of this
putative tumor suppressor gene through proteolysis is mediated by SCFSKP2
ubiquitin ligase complex. Adenovirus-mediated overexpression of SKP2 induced
ectopic down-regulation of p27 in LNCaP prostate carcinoma cells (Lu et al,
2002). This observation confirmed that SKP2 activity was the major determinant
of p27 levels in human prostate cancer cells. Based on in vitro studies, it is
believed that the overexpression of SKP2 might be one of the mechanisms
allowing prostate cancer cells to escape growth control mediated by p27.
Therefore, knocking out SKP2 function would be a logical novel approach to
fight prostate cancer. In another study, an adenovirus construct carrying p27
coding sequences Adp27(Kip1) was generated to assess whether the overexpression
of p27 has any affect on the prostatic tumor growth in vivo (Katner et al,
2002). Injection of Adp27(Kip1) vector reduced the growth of LNCaP tumor
xenografts in mice. This study supported the idea that Adp27(Kip1) can serve as
a potential therapeutic vector for the treatment of prostate carcinoma.
p14(ARF), encoded
by the human INK4a gene locus, is another tumor suppressor protein which is
frequently inactivated in human cancer. p14(ARF) has recently been implicated in
p53-independent cell cycle regulation and apoptosis. A replication-deficient
adenoviral construct carrying p14(ARF) coding sequence (Ad-p14(ARF)) was
generated in order to explore the pro-apoptotic function of p14(ARF) in
relationship to p53 function (Hemmati et al, 2002). Ad-p14(ARF) construct
induced apoptosis in p53/Bax-mutated DU145 prostate cancer cells and HCT116
cells lacking functional Bax expression. This study demonstrated that
overexpression of p14 through adenovirus vectors is sufficient to induce
apoptosis in p53- and bax-deficient prostate cancer cells. Prostate carcinoma
with p53 mutant phenotype represents a clear obstacle for irradiation therapy.
Ionizing radiation (IR) and adenoviral p53 gene therapy (Ad5CMV-p53) were
utilized individually as well as in combination in order to assess the
effectiveness of combined therapy for prostate cancer (Sasaki et al, 2001). In
this study, IR alone did not induce significant levels of apoptotic cell death
in DU145 and PC-3 cells. However, after combined therapy, the proportion of
apoptotic cells was greatly amplified in both of the cell lines tested.
Therefore, it was concluded that the observed synergistic effect might be
useful for the treatment of radio-resistant prostate carcinoma.
The loss of MMAC/PTEN
tumor suppressor gene expression is frequently detected in human tumors.
Survival signaling through the phosphatidylinositol-3 kinase/Akt pathway is
constitutively activated in cells lacking functional PTEN expression.
Therefore, the functional effect of MMAC/PTEN expression was examined in LNCaP
cells, which are devoid of a functional PTEN product (Davies et al, 1999).
Infection with an adenovirus construct driving the expression of MMAC/PTEN
resulted in a specific inhibition of Akt/PKB activation. This is consistent
with the phosphatidylinositol phosphatase activity of MMAC/PTEN. Compared to
adenovirus delivered p53 expression, MMAC/PTEN expression induced apoptosis in
LNCaP cells to a lesser extent. Interestingly, the growth suppression
properties of MMAC/PTEN were significantly greater than those accomplished with
p53. Moreover, Bcl-2 overexpression in LNCaP cells blocked both the adenovirus
mediated MMAC/PTEN- and p53-induced apoptosis, but it did not affect the
growth-suppressive properties of MMAC/ PTEN. This is consistent with the fact
that MMAC/PTEN may play multiple roles in the cell. Prostate cells were
infected with adenovirus vector carrying PTEN coding sequence in order to
determine if supplying PTEN function would sensitize these cells to various
apoptotic stimuli (Yuan and Whang, 2002). As predicted, adenovirus-mediated
PTEN delivery sensitized LNCaP prostate cancer cells to apoptosis through the
inhibition of constitutive Akt activation. Since PTEN G129E mutant lacking
lipid phosphatase activity was unable to sensitize cells to apoptosis, it was
concluded that the lipid phosphatase activity of PTEN was required for
apoptosis. The therapeutic effect of adenoviral delivery of MMAC/PTEN was
tested on both the in vitro and in vivo growth of PC3 human prostate cancer
cells (Davies et al, 2002). The in vitro growth of PC3 cells was repressed by
adenovirus expression of MMAC/PTEN via blocking of cell cycle progression.
Although this approach did not inhibit the tumor progression of orthotopically implanted
PC3 cells, a significant reduction was observed in the tumor size in vivo, in
addition to complete inhibition of metastases. Therefore, it was suggested that
MMAC/PTEN might play a role mostly in the regulation of the metastatic
potential of prostate cancer.
A considerable
fraction of prostate tumors display an alteration of Mxi1 expression, an
antagonist to c-Myc. This was confirmed by transgenic approaches in which
prostatic hyperplasia was observed in mice deficient for Mxi1. Mxi1-expressing
adenovirus (AdMxi1) was generated to study the ability of Mxi1 to act as a
growth suppressor in prostate tumor cells (Taj et al, 2001). Overexpression of
Mxi1 using adenovirus vectors in the DU145 prostate carcinoma cell line
resulted in growth arrest and decreased colony formation on soft agar. All
these studies emphasize that the modulation of tumor suppressor gene function
might be necessary for an optimum therapeutic response to fight against
prostate cancer.
VIII. Cell
adhesion molecules and anti-angiogenic approaches
Cell adhesion
molecules play major roles especially in metastasis of cancer cells. Therefore,
aberrant expression patterns of cell adhesion molecules are frequently
associated with poor prognosis. For instance, the expression of a well-known
cell adhesion molecule, C-CAM1, is downregulated during the early stages of
prostate carcinoma in an animal model (TRAMP) (Pu et al, 1999). C-CAM1 was
cloned into an adenovirus construct and its efficacy was tested both in vitro
and in vivo using PC3 xenograft murine model (Lin et al, 1999). AdC-CAM1
construct manifested a strong antitumoral activity on PC3 tumor cells grown in
nude mice. Therefore, selective use of cell adhesion molecules might be
beneficial for the treatment of prostate carcinoma. Moreover, combining
C-CAM1-based therapy with TNP-470, a potent angiogenesis inhibitor, induced
greater growth suppression on DU145 tumor xenografts than by either Ad-C-CAM1
or TNP-470 application alone (Pu et al, 2002).
Vascularization
of a solid tumor is required for cancer growth. Recently, preventing
vascularization through inhibition of angiogenesis was a popular target for
cancer gene therapy. For example, a 16-kDa prolactin protein (PRL) has
previously been shown to possess an antiangiogenic activity (Galfione et al,
2003). Not surprisingly, adenovirus delivery of PRL protein manifested a
significant antitumoral activity in vivo (Kim et al, 2003). In addition,
vascular endothelial growth factor (VEGF) receptor signaling is another
relevant pathway, which modulates the vascularization of newly growing tumors.
Interfering with such a signaling pathway might be valuable in controlling the
tumor growth. In fact, when fused to an Fc domain and cloned into the
recombinant adenovirus construct, the ligand-binding ectodomain of VEGF
receptor 2 (Flk1) manifested a considerable reduction in tumor growth induced
by a drastic decline in the microvessel density in SCID mice carrying human
LNCaP xenografts (Becker et al, 2002).
Growth factors
are needed for survival of cancer cells and molecular chaperones are required
for functional production of these molecules. A new member of the heat shock
protein family functioning as a molecular chaperone in the endoplasmic
reticulum was recently discovered and named as 150-kDa oxygen-regulated protein
(ORP150). Since prostate cancer cells exhibited an upregulation of ORP150
protein and VEGF, adenovirus delivery of an antisense ORP150 cDNA approach was
used to reduce angiogenicity and tumorigenicity through inhibition of VEGF
secretion. This approach indeed suppressed the growth of DU145 prostate
carcinoma cell line in a xenograft model (Miyagi et al, 2002).
IX. Replication
competent adenovirus vectors
Replication
competent adenoviral vectors provide powerful means to kill cancer cells
through cell lysis. Since they only replicate in tumor cells, the therapeutic
range is limited to cancer cells. Two replication-competent adenoviruses, CV706
and CV787, were generated in order to selectively destroy PSA producing
prostate cancer cells. It has been demonstrated earlier that prostate-specific
antigen (PSA)-selective replication-competent adenovirus variant CV706
specifically eliminated tumors in human prostate cancer xenografts in
preclinical models (Rodriguez et al, 1997). Since adenovirus E1A is known to be
a potent inducer of chemosensitivity and radiosensitivity through p53-dependent
and independent mechanisms, the potential radiosensitizing effects of CV706 on
prostate cancer cells were evaluated (Chen et al, 2001). The CV706 construct
demonstrated a synergistic antitumoral effect both on irradiated human prostate
cancer cells and tumor xenografts. Moreover, in order to investigate the safety
and the functionality of intraprostatic delivery of CV706 for the treatment of
patients with locally recurrent prostate cancer following radiation therapy, a
Phase I dose-escalation study was conducted (DeWeese et al, 2001). Results from
this study suggested that even at high doses, intraprostatic delivery of the
CV706 was relatively safe for patients and CV706 construct demonstrated high
therapeutic activity as reflected by the reduction in serum PSA. This was the
first clinical trial of a prostate-specific, replication-restricted adenovirus
for the treatment of prostate cancer. Another prostate-specific
replication-competent adenovirus carrying not one, but two, cell type specific
promoters (CV787) was constructed. This construct contained E1B gene driven by
the human prostate-specific enhancer/promoter and the adenovirus type 5 (Ad5)
the E1A gene under the control of prostate-specific rat probasin promoter. The
Ad5 E3 region was also conserved in the vector to improve the efficacy. A
single tail vein injection of CV787 eliminated LNCaP xenografts within 4 weeks
in nude mice (Yu et al, 1999). When the prostate cancer-specific adenovirus
CV787 was combined with chemotherapeutic agents like taxanes (paclitaxel and
docetaxel), a synergistic antitumoral effect was observed in mice carrying
human prostate cancer xenografts (Yu et al, 2001b).
Heat-inducible
gene expression is another approach used in the context of suicide gene
therapy. A recombinant adenovirus containing the CD-TK fusion gene controlled
by the human inducible heat shock protein 70 promoter (Ad.HS-CDTK) was
generated for this purpose. Heat application at 41oC for 1 hour induced
therapeutic gene expression from this vector. Despite the fact that the
Ad.HS-CDTK construct induced CD-TK expression in human prostate cancer cells, a
therapeutic benefit was not observed due to lower transduction efficiency of
tumors in vivo. Instead, a replication-competent, E1B-attenuated adenoviral
vector containing the hsp70 promoter-driven CD-TK gene (Ad.E1A+HS-CDTK) was
generated to increase CD-TK gene expression to achieve a therapeutic effect
(Lee et al, 2001). Contrary to replication incompetent Ad.HS-CDTK, replication
competent Ad.E1A+HS-CDTK construct yielded severe cytotoxicity and greater
levels of therapeutic index in the presence of prodrugs. This approach revealed
the beneficial effects of using replication competent virus complemented with a
heat inducible suicide gene therapy approach for prostate carcinoma.
X. Adenovirus
vectors with cell type specific and inducible promoters
Even though
adenovirus-mediated HSVTK suicide gene therapy approach manifested a
satisfactory toxicity profile in Phase I clinical trials, the toxicity studies
using adenovirus vectors were very restricted in numbers. However, it was known
that the promoter of choice might influence the level of toxicity. In order to
study the promoter effect on adenovirus mediated toxicity the mouse caveolin 1
promoter was cloned into the adenovirus HSV-tk vector (Adcav-1tk) because this
promoter was highly active in metastatic and androgen-resistant prostate cancer
cells (Pramudji et al, 2001). The efficacy of this vector for suicide gene
therapy was compared to those of AdHSV-tk vectors carrying either
cytomegalovirus (AdCMV-tk) or rous sarcoma virus (AdRSV-tk) promoters in mice
transplanted with mouse prostate cancer cells. Following GCV administration,
all the HSV-tk expressing vectors regressed the tumor growth in situ.
Interestingly, the efficacy of Adcav-1tk vector was much greater in terms of
inducing necrosis and microvessel density. In order to evaluate the toxicity
profile of adenovirus vectors carrying CMV, RSV or mouse caveolin
promoter-driven HSV-tk transgenes, these vectors were also injected
systemically into mice (Ebara et al, 2002). Adenovirus vectors with CMV and RSV
promoters, but not caveolin promoter, exhibited significant levels of liver
damage. These results suggested that the promoter selection greatly influences
the toxicity profile of adenovirus-mediated suicide gene therapy approach. In
order to increase the number of promoters available for prostate specific gene
expression, transgenic mice were generated expressing a reporter gene (SV40
Tag) directed by prostate secretory protein of 94 amino acids (PSP94) (Gabril
et al, 2002). PSP94 gene promoter/enhancer region directed SV40 Tag expression
exclusively in prostate leading to prostatic intraepithelial neoplasia and
eventually to high-grade prostate carcinoma. These studies suggested that this
PSP94 gene promoter/enhancer strategy could be employed for the treatment of
prostate carcinoma.
One conventional
way to limit the toxicity of virus mediated suicide gene therapy is to use cell
type specific promoters as suggested above. Although adenovirus vectors with
the native PSA enhancer and promoter (PSAP) provided prostate-specific
expression, lower transcriptional activity observed in prostate challenged its
use in prostate-targeted gene therapy. To improve the activity and specificity
of the prostate-specific PSA enhancer for gene therapy, various studies were
carried out by exploring the properties of the natural PSA control regions.
Chimeric PSA enhancer constructs were generated with tandem copies of the
proximal ARE elements and then inserted into adenovirus constructs
(Ad-PSE-BC-luc) (Wu et al, 2001). This construct was highly inducible with
androgens as shown by systemic administration into SCID mice carrying LAPC-9
human prostate cancer xenografts while retaining prostate specific gene
expression. Furthermore, the CreLoxP system was also utilized to enhance the
activity of PSAP. CD suicide gene therapy approach using adenoviral vectors
with CRELoxP augmented PSAP activity effectively inhibited subcutaneous LNCaP
tumor growth in nude mice (Yoshimura et al, 2002). In addition, hormone
refractory prostate cancer cells retain the expression of prostate-specific
membrane antigen (PSMA) and prostate-specific antigen (PSA). An adenovirus
construct with an artificial chimeric enhancer (PSES) composed of two modified
regulatory elements of PSA and PSMA genes (Ad-PSES-luc) was generated and
tested for its promoter activity for the treatment of prostate cancer (Lee et
al, 2002a). Systemic injection of Ad-PSES-luc construct into mice produced very
low levels of reporter gene expression in major organs. However, when injected
directly into prostate, only the prostate but not other tissues produced high
levels of reporter gene expression. These results encouraged the use of PSES
for the treatment of androgen-independent prostate carcinoma. Even though
prostate-specific antigen (PSA/hK3) provided prostate specific gene expression,
its expression displayed an inverse correlation with prostate cancer grade and
stage, giving reason to doubt its effectiveness for advanced stage of prostate
carcinoma. A new approach was developed in order to generate gene therapy
vectors targeting higher grades especially of prostate carcinoma. The human
glandular kallikrein 2 (hK2) is upregulated in an advanced form of prostate
cancer with a higher grade. Therefore the hK2 promoter was cloned into
adenovirus construct in combination with EGFP reporter gene (ADV.hK2-E3/P-EGFP)
in order to obtain preferential expression of EGFP in prostate cancer (Xie et
al, 2001a). Indeed ADV.hK2-E3/P-EGFP injection led to a robust but
tumor-restricted EGFP expression in subcutaneously generated LNCaP tumors.
These results showed that adenovirus constructs with the hk2
multienhancer/promoter driven therapeutic genes might be a powerful tool for
gene therapy of advanced prostate cancer.
Previous studies
have shown that the bone matrix protein osteocalcin is predominantly expressed
in prostate cancer epithelial cells, fibromuscular stromal cells and
osteoblasts. A conditional replication competent adenovirus vector carrying the
osteocalcin promoter driven early E1A gene (AdOCE1A) was generated to co-target
both prostate cancer cells and their surrounding stromal cells (Matsubara et
al, 2001). Both PSA-producing (LNCaP) and non-producing (DU145 and PC3) human
prostate cancer cell lines as well as human stromal cells and osteoblasts were
effectively killed by this recombinant virus in vitro. In addition a single
systemic intravenous injection of the AdOCE1A construct significantly destroyed
prostate tumor cells transplanted in SCID mice. This co-targeting strategy
appeared to have a broader effect compared to other recombinant constructs tested
on the preclinical models of human prostate cancer. These promising results
initiated first gene therapy trial (phase I) in which adenoviruses carrying the
osteocalcin promoter driven HSV-tk gene (AdOCHSVTK) were directly injected into
prostate cancer lymph node and bone metastasis (Kubo et al, 2003). The results
of this trial suggested that adenoviruses did not display any adverse effects
and the treatment was well tolerated in all patients. In addition, 63 % of the
patients had local cell death in treated lesions. Further studies are suggested
in order to assess the efficacy of this approach for androgen-independent
prostate carcinoma. A new treatment modality to enhance adenoviral replication
by vitamin D3 in androgen-independent human prostate cancer cells and tumors
was tested using a novel replication-competent adenoviral vector, Ad-hOC-E1,
carrying the human osteocalcin (hOC) promoter to drive both the early viral E1A
and E1B genes (Hsieh et al, 2002). While the replication properties of Ad-hOC-E1
vector were restricted to OC-expressing cells, vitamin D3 exposure further
enhanced viral replication by 10 fold. The growth of both androgen-dependent
and androgen-independent prostate cancer cells was suppressed by Ad-hOC-E1
infection, irrespective of the cells androgen responsiveness and PSA status.
This is in contrast to Ad-sPSA-E1 vector, which only replicated in
PSA-expressing cells with androgen receptor (AR). Ad-hOC-E1 injection inhibited
the growth of DU145 (an AR and PSA-negative cell line) tumor xenografts in
mice. Consequently, vitamin D3-enhanced Ad-hOC-E1 viral replication represented
an alternative for the treatment of localized or osseous metastatic prostate
cancer. Prostate specific antigen promoter (PSAP) and rat probasin (rPB)
promoter are currently employed to drive the therapeutic transgene expression
in prostate cancer cells. However, since these promoters require the binding of
androgen to androgen receptor for activation, they were only functional in
androgen-dependent prostate carcinoma cells. Because androgen refractory
prostate carcinoma cells lose the expression of androgen receptor along the
way, constructs with PSAP or rPB promoters are not useful for treating patients
with androgen-independent prostate carcinoma. In order to circurment this
problem, prostate specific promoters were modified so that they were activated
in response to the retinoids-retinoid receptor complex in place of the
androgen-AR complex. As a result, retinoid treated androgen-independent
prostate cancer cells were sensitized to HSVTK-ganciclovir gene therapy using
promoters responding to retinoids (Furuhata et al, 2003).
Apart from
promoters providing tissue specific gene expression, expression inducible
promoters were cloned into adenovirus constructs to control the onset and the
duration of gene expression. Tetracycline-inducible adenovirus vectors
expressing the cytokine interleukin-12 were successfully tested in an
immunotherapy model for prostate cancer (Nakagawa et al, 2001). Thus,
recombinant adenovirus vectors with tetracycline-inducible gene expression
opened up new avenues while improving the safety of viral vector administration
for cancer gene therapy. Limitation of cytotoxic gene expression only to tumor
cells is very much desired in adenovirus-mediated gene therapy approach for
cancer. Unfortunately, the expression levels of many tumor and tissue-specific
promoters are much lower than the constitutively active promoters. A complex
adenoviral vector was generated by fusing the tetracycline transactivator gene
to a prostate-specific ARR2PB promoter while placing a mouse FASL-GFP fusion
gene under the control of the tetracycline responsive promoter. This allowed
the joining of cell-type specificity with high-level regulation of transgene
expression (Rubinchik et al, 2001). The doxycycline regulated, ARR2PB driven
FASL-GFP vector generated higher levels of prostate-specific FASL-GFP
expression than FASL-GFP expression directed with ARR2PB alone, leading to
apoptosis in LNCaP cells. Systemic delivery of both the prostate-specific and
the prostate-specific/tet-regulated vectors was well tolerated in animals at
doses that were lethal for adenovirus vectors with CMV-driven FASL-GFP
expression. This approach improved the safety and efficacy of adenovirus-mediated
cytotoxic gene delivery for the treatment of prostate carcinoma.
The
prostate-specific adenovirus gene expression technology can also be used for
the identification of metastatic lesions of prostate cancer through the use of
non-invasive imaging. A prostate-specific adenovirus vector expressing a
luciferase reporter gene (AdPSE-BC-luc) and a charge-coupled device-imaging
system were employed for this purpose (Adams et al, 2002). A robust expression
from AdPSE-BC-luc construct was found in the prostate, especially in the
androgen-independent tumors. Furthermore, metastatic lesions in the lung and
spine with prostatic origin were identified successfully through repetitive
imaging over a three-week period after AdPSE-BC-luc injection into
tumor-bearing mice. These results demonstrate that adenovirus gene delivery
specific to the prostate can be coupled to a non-invasive imaging modality for
therapeutic and diagnostic strategies for prostate cancer.
XII. Adenovirus
vectors for vaccination and adjuvant gene therapy
CAR receptors and
MHC class I heavy chains are important mediators of adenovirus entry into tumor
cells. Contrary to the cell lines derived from other malignancies, down
regulation of CAR or MHC class I expression is relatively rare in both human and
murine prostate carcinoma cells. This brought the possibility of developing
vaccine strategies for prostate cancer based on the modification of prostate
cancer cells using recombinant adenovirus vectors (Pandha et al, 2003). The
expression of prostate-specific antigen (PSA) is highly restricted to prostatic
epithelial cells. In fact, 95 % of patients with prostate carcinoma express
PSA, making this antigen a good candidate for targeted immunotherapy. A
recombinant PSA adenovirus type 5 (Ad5-PSA) was generated in order to activate
PSA-specific T-cell response with the potential of eliminating prostate cancer
cells (Elzey et al, 2001). Ad5-PSA immunized mice displayed a PSA-specific
cellular immunity involving CD8+ T lymphocytes. This approach deterred subcutaneous
tumor formation with RM11 prostate cancer cells expressing PSA (RM11psa).
However, this did not affect the growth of existing RM11psa tumors. On the
contrary, Ad5-PSA administration followed by intratumoral injection of
recombinant canarypox viruses (ALVAC) encoding interleukin-12 (IL-12), IL-2,
and tumor necrosis factor-( effectively eliminated established RM11psa tumors.
Surgery is one of
the conventional treatment modalities used against solid tumors. Due to the
fact that minor residual tumors following surgical operation may result in
local recurrence, surgery is neither efficient nor plausible for the treatment
of metastatic disease. Although AdHSV-tk gene therapy followed by ganciclovir
administration has been evaluated extensively as a potential treatment modality
for numerous tumors, it has not yet been proven to achieve a complete cure on
its own. Prostate-derived tumor models were used to evaluate the effects of
AdHSV-tk gene therapy as an adjuvant to surgery (Sukin et al, 2001). Lung nodules
of prostate cancer cells were generated by intravenous injection of tumor cells
in order to evaluate systemic effects. Following resection of subcutaneous
tumors, AdHSV-tk was delivered to the resection site. Toxicity, local tumor
recurrence, survival, and lung nodule formation were evaluated in animals;
increased survival and decreased recurrence accompanied by no systemic toxicity
were observed. Adjuvant AdHSV-tk gene therapy resulted in a significant
reduction in lung nodules as well. This study suggested that AdHSV-tk gene
therapy might be beneficial as an adjuvant for patients undergoing surgical
treatment of cancer.
XIII. Current
progress to overcome rate-limiting steps in adenovirus-mediated gene therapy
for prostate carcinoma
The success of
adenovirus mediated gene therapy for prostate carcinoma is effected by several
factors including the level of expression of the receptor which facilitates the
entry of the viral vectors into the cells, penetration of transgenes to
surrounding tissues, and finally the expression of the delivered gene.
Enhancing these factors has been the focus of many laboratories working on
adenovirus-mediated gene therapy for prostate carcinoma. Although a limited
number of studies have been completed regarding these issues, effectiveness of
prostate cancer gene therapy will certainly benefit from the progress in this
field.
A. Receptor
abundance
The presence of
the coxsackie adenovirus cell surface receptor, CAR, is required for an
effective adenovirus infection of target cells. CAR expression patterns of
normal prostate and prostate carcinoma were compared using immunohistochemical
approaches in order to assess the feasibility of adenovirus mediated gene
therapy for prostate cancer (Rauen et al, 2002). While a robust membrane staining
for CAR was detected in the metastatic prostate specimens with higher Gleason
scores, just lumenal and lateral cell membrane staining were detected in the
benign prostate epithelia. Therefore, adenovirus mediated gene delivery should
be more effective for aggressive prostate tumors than it is for benign cases.
B. Penetration of
hybrid therapeutic transgenes to the surrounding tissue
Despite the fact
that adenovirus could transduce cells very efficiently in vitro, adenovirus
mediated gene delivery is restricted by the inefficient transduction of
surrounding cells for a given tumor. In order to overcome this obstacle, an
important intercellular transport protein named VP22, was first fused to the
therapeutic transgene of interest (p53 gene) and then cloned into adenovirus
vector (Roy et al, 2002). Infection of p53 negative human prostate cancer cells
(LNCaP) by this approach generated very efficient gene delivery of p53,
inducing apoptosis not only in the infected cells but also in the surrounding
uninfected cells.
C. Enhancement of
transgene expression through transcriptional regulation
Although the use
of prostate specific promoters is necessary to limit the transgene toxicity,
the low level of transgene expression directed by these promoters represents a
barrier to gene therapy. The observation, which led to the idea that
chemotherapeutics enhanced the transgene expression from viral promoters,
represented a new approach to overcome this barrier. Two recombinant adenovirus
constructs were used to deliver p21WAF-1/CIP1 and p53 protein c-DNA under the
control of cytomegalovirus promoter to the metastatic androgen independent
prostate cancer cells treated with chemotherapeutic agents docetaxel or
paclitaxel (Li et al, 2002b). Both chemotherapeutics appeared to enhance
adenovirus mediated transgene expression in androgen independent prostate
cancer cell lines. This increase in transgene expression was attributed to the
enhancement of CMV promoter activity rather than the increased viral uptake.
Therefore, the observed synergy of gene therapy with these chemotherapeutics
may become useful when the transgene expression is a limiting factor for the
treatment of the metastatic androgen independent prostate cancer. The possible
use of other chemotherapeutic agents and their effect on prostate specific
promoters should also be explored.
XIV. Summary of
clinical trials
There are 636
clinical protocols involving 3496 patients employed in gene therapy worldwide
as reported to the Journal of Gene Medicine website by the year 2002. 403
clinical studies (63.4 %) with regard to gene therapy for cancer were tested on
2392 (68.5 %) patients. Adenovirus was the vector of choice in 171 of these
protocols (27 %), and 644 patients (18.4 %) received the adenovirus vector for gene
therapy. 22 out of 171 clinical protocols were engaged in adenovirus mediated
gene therapies targeting the prostate only as summarized in Table 1. 13 of
these were reported to be in Phase I, 3 trials in Phase II and the rest (5)
were in Phase I/II. There is no Phase III clinical study reported using
adenovirus vectors targeting prostate yet. Some of the adenovirus mediated gene
therapy approaches were complemented either with radiotherapy or radical
prostatectomy. The percentage of the choice of gene therapy modalities
targeting prostate is provided in Figure 3. The use of selectively replicating
adenovirus constructs leads other approaches followed by suicide gene therapy.
This is partly because not long ago astonishing results were obtained with
selectively replicating adenovirus constructs in the preclinical animal models.
It is also interesting to note that two of these clinical trials utilize
suicide gene therapy in combination with the selectively replicating adenovirus
approach (Figure 3). No clinical studies have been carried out using the death
ligand-mediated gene therapy approach and adenovirus vectors up to date.
However we should not be surprised if such trials are being initiated and we
encounter some of these in the near future. Although preliminary results are
very encouraging from these clinical investigations, clear conclusions can be
drawn only upon completion of these studies.
Considering all
these preclinical and clinical studies, we concluded that great progress in
adenovirus mediated gene therapy for prostate carcinoma has been made within
the last 3 years. While the molecular mechanisms responsible for prostate
carcinoma are not fully understood, the effectiveness of gene therapy is still
quite amazing. As more data become available on the understanding of prostate
carcinoma, we anticipate that more effective treatment modalities will be
developed using adenovirus to target prostate cancer.
Table 1. A
summary of ongoing clinical trials of adenovirus mediated gene therapy
targeting prostate as of 2002. The data was collected from the Journal of Gene
Medicine web site ( HYPERLINK
"http://www.wiley.co.uk/genmed/clinical" www.wiley.co.uk/genmed/clinical)
and published with the permission from (John Wiley and Sons 2002.
CountryInvestigatorMode
of TherapyPhaseCanadaA. K. StewartImmunotherapy (IL-2)ICanadaJ. DanceyImmunotherapy
(IL-2)IUSAPeter T. ScardinoSuicide gene therapy (HSV-tk) + radiotherapyIUSASimon
J. HallNeo-adjuvant suicide gene therapy (HSV-tk) + radical prostatectomyIUSAArie
BelldegrunTumor suppressor gene therapy (p53)IUSAChristopher J. LogothetisTumor
suppressor gene therapy (p53)I/IIUSADov KadmonNeo-adjuvant suicide gene therapy
(HSV-tk) + radical prostatectomyIUSAJonathan W. SimonsSelectively replicating
adenovirus (CN706)IUSAThomas A. GardnerSuicide gene therapy (HSV-tk)IUSAJae Ho
KimSuicide gene therapy (CD/Tk) with selectively replicating adenovirus +
radiotherapyIUSAE. Brian Butler Suicide Gene Therapy (HSV-tk) + radiotherapyI/IIUSAJeffrey
R. GingrichNeo-adjuvant CDK inhibitor (p16) + radical prostatectomyIUSAMartha
K. TerrisSelectively replicating adenovirus (CV787) + RadiotherapyI/IIUSAGeorge
WildingSelectively replicating adenovirus (CV787)I/IIUSAAlan PollackTumor
suppressor gene therapy (p53) + radiotherapyIIUSAThomas A. GardnerSelectively
replicating adenovirus with osteocalcin promoter (Ad-OC-E1A)IUSADavid M.
LubaroffImmunotherapy (PSA)IUSABrian J. MilesImmunotherapy (IL-12) +
radiotherapyIUSATheodore L. DeWeeseSelectively replicating adenovirus (CV706)IIUSAEric
J. SmallSelectively replicating adenovirus (CV787) + chemotherapyIIUSASvend O.
FreytagNeo-adjuvant suicide gene therapy (CD/Tk) with selectively replicating
adenovirus + RadiotherapyIUSAJohn M. CormanSelectively replicating adenovirus
(CG7060) + radiotherapyI/II
Figure 3.
Adenovirus mediated clinical gene therapy modalities for prostate. The types of
clinical gene therapy modalities for prostate are represented as percentages in
a pie graph in order to better appreciate the contribution of each treatment
modality.
Acknowledgments
This work is
supported by Akdeniz University Scientific Research Project Administration
Division Grants (#2002.01.0122.06, #2002.01.0122.07 and #2002.01.0200.005 to
Dr. Salih Sanlioglu).
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Sanlioglu
Sanlioglu et al:
Adenovirus mediated gene therapy for prostate carcinoma
Gene Therapy and
Molecular Biology Vol 7, page PAGE 113
PAGE
118
PAGE
113
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