Comparative evaluation of survivin, midkine, and CXCR4 promoters for transcriptional targeting of glioma gene therapy

Abstract

Transcriptional targeting is a key strategy to enhance therapeutic efficacy of gene therapy applications. In the context of oncolytic virotherapy, transcriptional promoter elements are used from genes that are over expressed in a variety of malignant cancers. In the present study, we examined the feasibility of transcriptional targeting to glioma cells by comparing the activity of survivin, midkine, and CXCR4 tumor-specific promoters.
To evaluate the expression level of several glioma related genes, we performed quantitative RT-PCR analyses on samples obtained from cell lines and patients. To determine specific level of gene expression mediated by selective promoter elements, we measured luciferase expression in glioma samples transduced with replication deficient adenoviral vectors. Finally, we incorporated the optimal promoters into a conditionally replicative adenoviral vector, CRAd-5/3, and examined the cytopathic effect in vitro.
The survivin promoter demonstrated the highest level of mRNA expression in primary tumor samples and cell lines. Transcriptional targeting was confirmed by infection of glioma cells with an adenovirus expression vector containing a surviving-driven luciferase reporter gene. Of the tested promoters, minimal level of survivin activity was detected in normal human liver and brain. A novel vector, CRAd-survivin5/3, with E1a under the control of the survivin promoter, exhibited enhanced cytopathic effect in vitro.
Our data demonstrate that the survivin promoter element is very active in glioma samples and has low activity in normal human brain and liver. A novel oncolytic virus, CRAd-survivin-5/3, was effective against a panel of glioma cell lines in vitro. Our results suggest that employing the survivin promoter element in the context of CRAd-5/3 may present a new opportunity for the development of glioma specific oncolytic vectors.

Full text

©2007 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
Research Paper
Comparative Evaluation of Survivin, Midkine and CXCR4 Promoters
for Transcriptional Targeting of Glioma Gene Therapy
[Cancer Biology & Therapy 6:5, 679-685, May 2007]; ©2007 Landes Bioscience
Ilya V. Ulasov1
Angel A. Rivera2
Adam M. Sonabend1
Lisa B. Rivera2
Ming Wang2
Zeng B. Zhu2
Maciej S. Lesniak1,*
1Division of Neurosurgery; The University of Chicago; Chicago, Illinois USA
2Division of Human Gene Therapy; Departments of Medicine, Pathology, Surgery;
The Gene Therapy Center; University of Alabama at Birmingham; Birmingham,
Alabama USA
*Correspondence to: Maciej S. Lesniak; The University of Chicago; Division of
Neurosurgery; 5841 S. Maryland Avenue; MC 3026; Chicago 60637 Illinois USA;
Tel.: 773.834.4757; Fax: 773.834.2608; Email: mlesniak@surgery.bsd.uchicago.edu
Original manuscript submitted: 11/24/06
Manuscript accepted: 02/03/07
Previously published online as a Cancer Biology & Therapy E-publication:
http://www.landesbioscience.com/journals/cbt/article/3957
KEY WORDS
adenovirus, CXCR4, glioma, midkine,
survivin, promoter
ACKNOWLEDGEMENTS
This study was supported by a grant from the
National Institute of Neurological Disorders
and Stroke (NINDS/NIH) 5K08NS046430
(M.L.) and the American College of Surgeons
(M.L.).
ABSTRACT
Objective: Transcriptional targeting is a key strategy to enhance therapeutic efficacy
of gene therapy applications. In the context of oncolytic virotherapy, transcriptional
promoter elements are used from genes that are over expressed in a variety of malignant
cancers. In the present study, we examined the feasibility of transcriptional targeting to
glioma cells by comparing the activity of survivin, midkine, and CXCR4 tumor‑specific
promoters.
Methods: To evaluate the expression level of several glioma related genes, we
performed quantitative RT‑PCR analyses on samples obtained from cell lines and patients.
To determine specific level of gene expression mediated by selective promoter elements,
we measured luciferase expression in glioma samples transduced with replication
deficient adenoviral vectors. Finally, we incorporated the optimal promoters into a con‑
ditionally replicative adenoviral vector, CRAd‑5/3, and examined the cytopathic effect
in vitro.
Results: The survivin promoter demonstrated the highest level of mRNA expression in
primary tumor samples and cell lines. Transcriptional targeting was confirmed by infec‑
tion of glioma cells with an adenovirus expression vector containing a survivin‑driven
luciferase reporter gene. Of the tested promoters, minimal level of survivin activity was
detected in normal human liver and brain. A novel vector, CRAd‑survivin5/3, with E1a
under the control of the survivin promoter, exhibited enhanced cytopathic effect in vitro.
Conclusions: Our data demonstrate that the survivin promoter element is very active
in glioma samples and has low activity in normal human brain and liver. A novel
oncolytic virus, CRAd‑survivin‑5/3, was effective against a panel of glioma cell lines
in vitro. Our results suggest that employing the survivin promoter element in the context
of CRAd‑5/3 may present a new opportunity for the development of glioma specific
oncolytic vectors.
INTRODUCTION
Glioblastoma multiforme (GBM) represents one of the most aggressive forms of
primary brain tumors. Even after clinically available treatments, including surgery,
radiotherapy, and chemotherapy, the median survival remains less than two years.1
Clearly, novel therapies for brain cancer are required in order to make an impact on this
devastating disease. Gene therapy and virotherapy constitute a novel therapeutic approach
for the treatment of glioblastoma. In gene therapy approaches, a therapeutic gene for
mutation compensation, immunopotentiation, or prodrug activation is transferred.2-7
In virotherapy, tumor cell killing is achieved by oncolysis; that is, virus replication
induced cell killing.8-10 Both of these therapeutic interventions allow for specific anti-
tumor effects via molecular targeting strategies that exploit tumor markers.
At present, one of the most promising gene delivery vehicles is the recombinant adeno-
viral (Ad) vector.9,11 Whereas adenoviral vectors are understood to exhibit superior levels
of in vivo gene transfer compared to some of the available alternative vector systems, their
present level of efficiency in clinical trials may nonetheless be suboptimal for cancer gene
therapy and virotherapy applications. Various approaches have been developed to enhance
the transcription selectivity of current vector systems for tumor cells, thereby limiting
ectopic expression in non-tumor cells and treatment-associated toxicities. Transcriptional
targeting strategies employ the use of tissue specific promoters (TSP) to restrict transgene
expression or viral replication to tumor cells. The ideal TSP for glioma would exhibit
the widest differential between ‘tumor on/brain and liver off’ expression profiles, which
is fundamental for ablation of normal brain toxicity and potential liver toxicity from
ectopically localized adenovirus.
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Several candidate TSP genes have been found to be upregulated
in glial tumors, specifically: the chemokine C-X-C motif receptor 4
(CXCR4), the inhibitor and apoptosis protein (IAP) survivin, and the
growth factor midkine.9,12-14 Mutations in these genes play a major
role in cancer by producing anti-apoptotic activity or enhancing
survival or growth signals. To combine the benefits of adenoviral gene
therapy and specificity of these TSPs for glioma, we designed repli-
cation deficient adenoviral vectors that incorporate these promoters
upstream of the luciferase reporter gene. These viruses were tested in
vitro in glioma cell lines as well as in primary human glioma tissue.
The viruses were also tested in normal human brain and liver.
Although we have previously shown the utility of survivin in glioma
gene therapy,15 the three different promoters have not been system-
atically compared and explored in glioma. Our study thus provides
valuable new information for glioma adenoviral based gene therapy
and virotherapy with respect to the most efficient transcriptional
targeting strategy. Moreover, it provides a compelling argument for
further preclinical development of CRAd-survivin-5/3, a novel onco-
lytic virus that utilizes the survivin promoter and contains a chimeric
fiber protein encompassing adenovirus serotype 3 knob, which binds
to CD46 expressed on malignant glioma.16
MATERIALS AND METHODS
Cell lines and tissue specimens. Human glioma tumor cell
lines A172, U87MG, U373MG and U118MG were purchased
from American Type Culture Collection (ATCC, Manassas, VA).
Kings and No. 10 cells were purchased from the Japanese Tissue
Tumor Bank (Tokyo, Japan). A172, U87MG, U373MG and
U118MG cells were maintained in minimal essential medium
(MEM) (Mediatech, Herndon, VA) supplemented with 10% (v/v)
fetal bovine serum (FBS, HyClone, USA), 2 mM L-glutamine,
penicillin (100 IU/ml), and streptomycin (100 mg/ml). Kings
and No. 10 cells were maintained in RPMI medium (Gibco, Life
Technologies, USA) supplemented with 10% (v/v) FBS, 2 mM
L-glutamine, and penicillin/streptomycin. All cells were incubated at
37˚C in an atmosphere containing 5% CO2.
Human brain tumor specimens were obtained from patients
diagnosed with glioblastoma multiforme (WHO grade IV) under an
Institutional Review Board approved protocol. Primary samples were
reviewed by a pathologist to confirm that all tumors were GBMs.
A total of five GBM tumor specimens designated as T5, T13, T15,
T16 and T17 were used.
Human brain and liver samples (n = 3) were obtained from
patients undergoing a craniotomy or after liver transplantation.
All specimens obtained from patients were immediately used in
the experiments. To generate slice organ cultures, the tissue was
serially dissected into 0.5 mm-thick slices using the Krumdieck tissue
slicer17 (Alabama Research Development, Munford, AL). Next, the
tissue was cultured in 6-well plates in RPMI medium supplemented
with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, and
5 mg/ml insulin. Cultures were maintained at 37˚C in a humidified
atmosphere of 95% air and 5% CO2 under continuous shaking.
Three tissue slices were examined per group.
Adenoviral vectors. The following recombinant adenoviral (Ad)
vectors were constructed in Division of Human Gene Therapy
(UAB) and used in this study: reAdGL3CMV, reAdGL3MK,
reAdGL3Survivin, and reAdGL3CXCR4. These vectors contain
the pGL3 plasmid Ad backbone and contain a luciferase expression
cassette under the human cytomegalovirus (CMV), midkine (MK),
survivin (SURV), or chemokine C-X-C motif receptor 4 (CXCR4)
promoter.18
The AdWT, CRAd-5/3 and CRAd-survivin-5/3 replication-
competent vectors were described before.19,20 CRAd-CXCR4-5/3
was created according to the scheme utilized for the production of
CRAd-survivin-5/3 vector, but instead of a shuttle plasmid carrying
the survivin promoter, we utilized a shuttle plasmid containing
CXCR4. All vectors were rescued in A549 cells. Viral particles were
purified on CsCl gradients by standard methods, and the concen-
tration of viral particles was determined by measuring absorbance
at 260 nm using a conversion factor of 1.1 x 1012 viral particles/
absorbance unit.
Isolation of RNA and real‑time quantitative RT‑PCR. Total
cellular RNA was extracted from cells with the RNeasy mini prep
kit (Qiagen, Ca, USA) and treated with DNAase I (Gibco, Life
Technologies, USA) for 30 min. RT-PCR products from midkine,
CXCR4, CMV and SURV were used for standard curve. RNA (1 mg)
isolated from samples was used in a one-step reverse transcription
PCR. The reaction was done using Gene Amp RNA PCR core kit
(Applied Biosystems, Inc., Foster City, CA). All primers and probe
set were design by the Primer Express software and synthesized by
Applied Biosystems, Inc. The primer and probe sequences used to
amplify and detect transcript were as follows (5'-3'): SURV sense
TGG AAG GCT GGG AGC CA; SURV antisense, GAA AGC
GCA ACC GGA CG; probe 6FAM-TGA CGA CCC CAT AGA
GGA ACA TAA AAA GCA T-TAMRA. Oligonucleotides for ampli-
fication CXCR4 gene were (5'-3'): CXCR4 sense, CTT CCC TTC
TGG GCA GTTGA; CXCR4 antisense, ACA TGG ACT GCC
TTG CAT AGG; and probe 6FAM-CCG TGG CAA ACT GGT
ACT TTG GGA ACT-TAMRA. The sequences to amplify midkine
were forward primer CAA TGC TCA GTG CCA GGA GA, reverse
primer TGG CTT TGG CCT TTG CTT T and probe 6FAM-CAT
CCG CGT CAC CAA GCC CTG-TAMRA. The primers to amplify
CMV gene were described before.18 A standard curve was generated
using serial 10-fold dilutions for quantification by reverse transcrip-
tion-PCR (RT-PCR). The PCR reaction included denaturation
(94˚C, 5 min) followed by 29 cycles, each consisting of denaturation
(94˚C, 1 min), annealing (60˚C, 1 min), and extension (72˚C,
2 min) with a final extension phase (10 min). The PCR reaction was
performed on a LightCycler system (Roche Molecular Biochemicals,
Model 2400). Reactions were incubated at 50˚C for 2 minutes,
60˚C for 30 minutes, 95˚C for 5 minutes, and then subjected to
40 cycles at the following conditions: 94˚C for 20 seconds and 60˚C
for 1 minute. Data was analyzed with LightCycler software. Each
data point was repeated two times. Quantitative values were obtained
from the threshold PCR cycle number (Ct), where the increase in
signal associated with an exponential growth of PCR product became
detectable. The relative mRNA levels in each sample were normalized
to GAPDH content.
To detect E1A expression, cells were cultured and infected as
previously described.21 At either 24 or 48 hours post infection, cell
culture media was removed, and the remaining cells were collected
by scraping into PBS. This solution was centrifuged, and supernatant
was removed. RNA obtained from the pelleted cells was stabilized
immediately using RNAlater reagent (Qiagen) and stored at -80˚C
until extraction. RNA was isolated using the standard protocol
provided by Qiagen for RNAeasy isolation. Contaminating DNA
was removed by DNase digestion (Qiagen). TaqMan primers and
probes were designed by Primer Express 1.0 software and synthesized
by Applied Biosystems. The sequences to amplify adenovirus genes
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Glioma Promoters for Transcriptional Targeting
were as follows: E1A forward, 5'-AACCAGTTGCCGTGAGAGT
TG-3'; E1A reverse, 5'-CTCGTTAAGCAAGTCCTCGATACA-3';
E1A probe, 5'-CACAGCCTGGCGACGCCCA-3'.21 All real-time
PCR reactions were performed under the same conditions as
described above.
Luciferase assays. For luciferase assay, 5 x 104 cells/well were
seeded onto 24-well plates 24 hours before infection. The next day,
cells were infected with adenovirus vectors at 100 vp/cell or mock
infected for 1 hour at 37˚C in fresh medium. After infection, the
cells were washed and fresh medium was added. At 48 hours after
infection, the medium was carefully removed and the cells were
harvested and luciferase activity was assayed in the cell lysates using a
standard luciferase expression protocol (Promega Wisconsin, USA).
Luciferase expression detected in glioma cells was normalized to
activity of human CMV promoter. To detect luciferase activity of
adenovectors in human samples, each group of three slices containing
a 250 mm piece of tissue was infected with 500 vp/cell by adding
virus in media containing 2% FBS. After 1 hour of virus infection,
the media was aspirated and media containing 10% FBS was added.
Forty-eight hours later, the tissues were harvested and luciferase
activity was assayed in the tissue homogenates using a standard
luciferase expression protocol presented by vendor (Promega). All
results are presented as ratio of luciferase activity per mg of total
protein. Data was recorded using a Modulus Luminometer (Turner
Biosystems, USA).
Detection of CRAd induced cytotoxicity. The in vitro cyto-
toxic effect of AdWT, CRAd-5/3, CRAd-CXCR4-5/3 and
CRAd-SURV-5/3 vectors was evaluated in a panel of glioma cell
lines. U373MG, U87MG, No.10 and U118MG cells were plate
in 24 well plate at a density of 50,000 cells per well. Twenty-four
hours later, the cells were infected for 1 hour with 300 mL of infec-
tion medium containing Ad vectors at doses of 1000, 100, 10, 1 and
0.1 vp per cell. Then the infection medium was replaced with the
appropriate complete medium. After 10 days, the cells were fixed
with 10% formaldehyde and stained with 1% crystal violet in 70%
ethanol solution.
Statistical analysis. All experimental determinations were
performed in triplicate. The CMV, CXCR4, SURV and MK mRNA
activity in tumors vs. normal tissue was compared using the Student’s
t test. In all analyses, p < 0.05 was considered statistically significant.
RESULTS
Survivin, CXCR4 and midkine mRNAs are overexpressed in
brain tumors compared to normal tissue. To investigate expression
level of the CXCR4, midkine, and survivin transcripts, we designed
specific real-time quantitative RT-PCR primers and probes recog-
nizing the expressed sequences of the human mRNAs. As shown in
Figure 1A, quantitative RT-PCR analysis of target transcripts in six
glioma cell lines of different morphological types revealed that all of
the glioma cell lines tested expressed detectable levels of CXCR4,
midkine, and survivin mRNAs. Among them, two glioma cell lines
(Kings and No. 10) demonstrated nearly the same pattern of expres-
sion characteristic of normal human brain tissue. In addition, four
of the six cell lines (A172, No. 10, Kings and U118MG) expressed
similar levels of midkine mRNA expression. In contrast to these
results, three glioma cell lines (U87MG, U118MG and U373MG)
showed increased expression of CXCR4 mRNA (26.97-fold to
63.21-fold) and in particular, survivin mRNA (158-fold to 672-fold)
as compared to normal human brain tissue (p < 0.05). The U87MG
and U373MG cell lines also showed significantly decreased expres-
sion of midkine mRNA (8.4 and 5.5-fold, p < 0.05) compared to
normal human brain tissue.
To detect expression of CXCR4, midkine, and survivin mRNAs
in primary tissues, we isolated total RNA from five GBM patients.
Quantitative RT-PCR revealed inter-individual variation in expres-
sion of all target mRNAs in these tissues (Fig. 1B). However, survivin
mRNA was significantly over-expressed in all primary gliomas tested
compared to matched normal human brain tissue (p < 0.05). Indeed,
the median difference in expression of survivin mRNA between
tumors and matched normal controls was 10,000-fold increased.
Four of five tumors samples analyzed also showed increased amounts
of CXCR4 mRNA (10–1000-fold, p < 0.05) compared to normal
human brain sample.
Level of luciferase expression driven by midkine, CXCR4 and
survivin promoters correlates with mRNA expression. The tran-
scriptional activities of CXCR4, survivin, and midkine promoters
driving luciferase expression in recombinant Ad vectors were evalu-
ated in the glioma cell lines (Fig. 2). In this experiment, the cell lines
were infected with the relevant adenoviral vectors of 100 vp/cell.
Figure 1. Results of survivin, CXCR4, and midkine mRNA expression in
glioma cell lines (A) and primary tumors (B). cDNAs were prepared from
total RNA extracted from each sample. The expression levels were deter‑
mined by quantitative RT‑PCR using equal amount of cDNA. Results of mRNA
expression were normalized to the GAPDH. All samples were determined in
duplicates twice (p < 0.05).
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AdCMVLuc was used to standardize for varying transduction
efficiencies between cell lines, and promoter activities were there-
fore plotted as percentage of cytomegalovirus (% CMV activity)
driven luciferase expression. In the glioma cell lines tested, each of
the candidate promoters demonstrated variable relative luciferase
activity between the different cell lines. The Ad vector containing
the survivin promoter, however, showed a higher relative luciferase
activity compared to the other candidate promoters in all six cell lines
tested. These results suggest that the survivin promoter is promising
for transcriptional targeting in glioma cell lines.
In primary slices (Fig. 2B), the survivin-driven vector demon-
strated the highest level of luciferase expression. These results support
the data obtained from mRNA expression in passaged cell lines.
Status of survivin, CXCR4 and midkine expression in normal
tissues. A key limitation for the use of a systemic adenoviral gene
therapy or virotherapy is the potential toxicity to non-target organs.
To investigate the specificity of expression of these candidate gene
promoters in glioma, we next compared the mean level of gene
expression in human liver, given the tropism of adenovirus for this
organ.22 Each of the candidate promoters was assessed for transgene
expression in fresh-cut human liver tissue and normal brain slices.
As shown in Figure 3A, transgene expression induced by CXCR4
promoter was similar in liver to that induced by the CMV promoter.
Of note, the survivin and the midkine promoters demonstrated the
lowest transgene expression (<1% of CMV activity).
To further evaluate the specificity of the candidate promoters
for potential use in localized gene therapy, normal human brain
(Fig. 3B) was infected with the different transcriptionally targeted
adenoviral vectors. In this experiment, tissue slices were transduced
with the Ad vectors and 48 hours later, transduction levels were
detected by luciferase expression. These results indicate that the
expression levels for all tested promoters were less than 2% relative
to the CMV promoter in normal brain tissues. These data underline
the previous results indicating that these promoters are a promising
selective tool for transcriptional targeting of glioma.
CRAds induce cytopathic effect in human glioma cells propor‑
tionate to the promoter activity detected by qRT‑PCR. Up to
this point, our results suggested that the survivin promoter may be
an optimal TSP for glioma therapy, with CXCR4 showing some
evidence of favorable activity. Based on these findings, we incorpo-
rated either the survivin or CXCR4 promoter into a novel oncolytic
vector, CRAd-5/3. This is a chimeric vector which contains the Ad5
backbone and Ad3 knob and binds to CD46 which is expressed on
malignant brain tumors.16 To investigate whether human survivin or
Figure 2. Transcriptional activity of promoter units detected by luciferase
assay. Human glioma cell lines (A) or tumor slices obtained from patient
specimen (B) were infected with replication deficient adenoviruses. Forty‑eight
hours later, luciferase detection was performed. Results are presented as
percent of CMV activity.
Figure 3. Ex vivo analysis of adenovirus replication in human liver slices (A)
and normal human brain (B). Tissue slices were incubated with 500 vp/slice
of each virus for 1 hr and then fresh media was added. Forty‑eight hours
later, luciferase expression was measured. Each point represents the mean
of triplicates from two independent experiments. Values for each vector are
as percent of CMV activity ± SD from three experiments.
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CXCR4 promoter can accelerate a cytopathic effect (CPE) in infected
tumor cells, CPE assays were done on U373MG, No. 10, U118MG,
U87MG human glioma cells. As shown in Figure 4, CRAd-SURV-5/3
infected U373MG and U87MG cells were completely lysed at
a 1000 vp/cell whereas No.10 and U118MG cells were killed at
100 vp/cell. In U373MG cells, killing activity was observed only for
AdWT and CRAd-CXCR4-5/3 vectors. Our results demonstrate
that incorporation of promoter elements into adenoviral genome
increases transcriptional control of E1A adenoviral region and
induces oncolysis.
Next, we infected U373MG, No.10, U118MG and U87MG
human glioma cells with AdWT, CRAd-5/3, CRAd-CXCR4-5/3
or CRAd-SURV-5/3 at 10 vp/cell. Forty-eight hours post-
infection, quantitative RT-PCR analysis demonstrated that the
CRAd-SURV-5/3 vector exhibited superior expression of E1A copy
numbers in U118MG (775.28 ± 12.57 fold), U87MG (454.3 ±
23.61 fold) and No.10 (557.8 ± 59.45) infected cells vs. AdWT
(p < 0.05, Fig. 5). This result indicates that combination of trans-
ductional approach (3 knob modification) and a transcriptional
modification (incorporation of survivin promoter) results in both
enhanced replication and enhanced cells lysis.
DISCUSSION
The therapeutic efficacy of virotherapy relies on a vector’s ability
to successfully target, transduce, and replicate in GBM. In order to
specifically replicate in tumor tissue, the adenovirus must posses a
TSP element in its genome which responds to the specific cellular
cues of tumor cells to mediate its replication. Unfortunately, the
number of available promoters with glioma specific activation
properties is very limited. While a number of TSP elements have
been tested in the context of GBM, there has been no comparative
analysis of these promoters in order to determine the optimal agent
for further preclinical and clinical development. This gap in knowl-
edge prompted us to compare three of the most widely utilized TSP
in glioma-CXCR4, midkine, and survivin.
CXCR4 is a chemokine which was originally described in the
context of providing migrational clues for leukocytes. However,
CXCR-4 has been found to be upregulated on malignant glioma.9,23-26
In a seminal article, Zhou et el., showed that CXCR4 on glioma
lines is a signaling receptor in that its agonist, stromal cell-derived
factor-1 (SDF-1; CXCL12), produced rapid phosphorylation of
mitogen-activated protein kinases.9 Furthermore, SDF-1 induced the
phosphorylation of Akt (protein kinase B), a kinase associated with
survival, and prevented the apoptosis of glioma cells. Most recently,
Ehtesham et al., have shown that invasive populations of glioma
cells overexpress CXCR4 at the message and protein levels, and that
this expression ranges from 25- to 89-fold higher than that found in
noninvasive tumor cells.25 Furthermore, neutralization of CXCR4
significantly impaired the in vitro invasive capacity of malignant glial
cells. Taken together, these findings underscore the importance of
CXCR4 as a potential therapeutic target for the treatment of invasive
glioblastoma.
The midkine promoter has been widely evaluated as a valuable tool
for cancer gene therapy as this regulatory sequence has been shown
selectively active in many malignancies including gliomas.27-34 The
activity of this promoter might be down-regulated by p53-dependent
pathways but further evidence is needed to elucidate the mechanism
of this phenomenon.32 In the case of glioma, midkine promoter
activity was shown to be two times higher in midkine-positive cells
than in midkine-negative primary normal brain cells.35 For this
reason, a midkine promoter-based CRAd (Ad-MK) was developed.27
This vector showed strong oncolytic activity in midkine-positive
glioma cells but did not exhibit cytotoxicity in midkine-negative
primary normal brain cells. When tested in an in vivo experiment,
Ad-MK eradicated midkine-positive glioma xenografts.
Finally, survivin is a novel member of the inhibitor-of-apoptosis
family which appears to be inappropriately expressed in human
cancers. The survivin promoter appears to be a promising tumor-
specific promoter in the context of human melanoma, breast cancer,
and glioma.27,36-38 Most recently, survivin has been found to be over-
expressed in up to 79% of astrocytic tumors.39-41 The expression of
this gene correlates with grade and is present in 90% of glioblastomas.
The activity of this promoter is enhanced by hypoxia,42 commonly
found in rapidly growing tumors like high grade gliomas. Survivin
seems to play an important role in the oncogenesis and progression
of these tumors.39,43,44 This is suggested by its expression pattern and
by the fact that patients with survivin-positive astrocytic tumors have
significantly shorter overall survival times compared with patients
who have survivin-negative tumors.39 The promoter has already
been proven effective for transgene expression in lung cancer.45 We
have recently shown that the incorporation of this promoter into the
adenoviral E1a region is responsible for enhanced viral replication
and an enhanced oncolytic effect in malignant glioma.15
The results of our comparative study indicate that of the tested
TSPs, the human survivin promoter is elevated in both passaged
glioma cells lines as well as primary tumor tissue. In fact, in three
of the six passaged glioma cells lines, we observed at least a 1,000
fold increase in survivin mRNA expression vs. normal human brain.
Similar results were observed in primary tissue, where the level of
survivin mRNA expression was 10,000 fold increased over normal
human brain. When utilized as a TSP, survivin increased transgene
Figure 4. Cell killing of human glioma cells by CRAd‑CXCR4‑5/3 and
CRAd‑SURV‑5/3 vectors. No.10, U373MG, U87MG or U118MG human
glioma cells were infected with AdWT, CRAd‑5/3, CRAd‑CXCR4‑5/3 or
CRAd‑SURV‑5/3 at indicated vp/cell. Noninfected cells served as control.
Ten days later, cells were stained with crystal violet.
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expression in all of the passaged (at least 10-fold) and primary glioma
(10–100 fold) tumor tissue. This finding was significant not only
with respect to control but also with respect to CXCR-4 and midkine
promoters. This study clearly establishes survivin as an optimal TSP
for glioma gene therapy.
Of note, we also examined the specificity of our promoters in
the context of human liver and normal brain. Adenoviral vectors
exhibit enhanced tropism for liver tissue, with hepatotoxicity
being a limiting side-effect of adenoviral based gene therapy.46 We
detected very low levels of survivin promoter activity in normal liver
(<1% of CMV activity) and human brain (<1% CMV activity).
Taken together, these studies establish the specificity of survivin
for malignant brain tumors and suggest a preclinical safety profile
which warrants further analysis and testing of these promoters in
in vivo experimental schemas. The improved efficacy of transgene
expression in malignant brain tumors along with improved safety
index in normal human liver and brain validate the use of survivin
in transcriptional targeting strategies to restrict transgene expression
and viral replication to tumor cells.
In conclusion, we have performed the first comparative study of
three promoters which are widely utilized in glioma gene therapy.
Our results suggest that the survivin promoter is an ideal TSP for
development in both gene therapy and virotherapy glioma clinical
trials. A combination strategy involving transcriptional and trans-
ductional modification of an adenovirus led us to the development
of a novel oncolytic vector, CRAd-SURV-5/3. When tested against a
panel of glioma cell lines, the virus preferentially killed glioma tumor
cells in vitro and provided a “proof-of-principle” with respect to the
use of TSP promoter elements in glioma gene therapy.
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