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(CANCER RESEARCH 56. 3038-3041.
July I. 1996|
Antitumor Effects of an Adenovirus Expressing Antisense Insulin-like Growth
Factor I Receptor on Human Lung Cancer Cell Lines
Choon-Taek Lee,1 Shan Wu, Dmitry Gabrilovich,
David P. Carbone2
Hailei Chen, Sorena Nadaf-Rahrov,
lija F. Ciernik, and
Hatntm Center for Therapeutic Oncologv Research. University of Texas Southwestern Medical Center. Dallas. Texas 75235
physically smaller than normal), indicating that relatively normal
development and tissue differentiation can occur in the absence of
IGF-Ir (10). These findings suggest a potential basis for tumor selec
ABSTRACT
Insulin-like growth factors (IGFs) are often essential for the mainte
nance of the malignant phenotype, and in lung cancer the IGF-I receptor
(IGF-Ir) is often expressed at high levels. Stable transfection of antisense
plasmids expressing the first 300 bp of the IGF-Ir reduces the tumorige-
tivity in therapeutic applications.
The stable introduction of an antisense plasmid or the use of
antisense oligonucleotides presents a variety of obstacles in potential
clinical gene therapeutic strategies. Retroviruses are hampered by the
difficulty of production and their inefficiency of transduction. Adenoviruses may represent excellent vectors for the introduction of IGF-Ir
antisense constructs into tumors. Adenoviruses are highly infective for
the actively dividing, slowly dividing, and nondividing tumor cells
that often coexist in solid tumors, and they express high levels of the
transduced gene (11, 12).
The IGFs and their receptors are very important in lung develop
ment and the growth of cells in the respiratory system (13). In many
lung cancer cell lines, IGF-I and IGF-Ir can mediate autocrine pro
liferation (14, 15). Therefore, in this study, we constructed an adeno
virus expressing antisense IGF-Ir (Ad-IGF-Ir/as) and sought to deter
mine its effectiveness in the reduction of IGF-Ir expression and in the
nicity of a variety of tumor cell lines and has been reported to induce
systemic antitumor effects on established, non-gene-modified tumors in
animal model systems. We have
antisense IGF-Ir (Ad-IGF-Ir/as)
tions into a clinical therapeutic
IGF-Ir/as (at a multiplicity of
constructed an adenovirus expressing an
in an attempt to develop these observa
approach. A single transduction by Adinfection of 10:1) decreased the IGF-Ir
number by about 50% in human lung cancer cell lines NCI H460 and
SCC5, as measured by an I25l-labeled IGF-I competitive binding assay.
After the transduction
of these human lung cancer cell lines by Ad-IGF-
Ir/as, the soft agar clonogenicity was reduced by 84%. The i.p. treatment
of nude mice bearing established i.p. NCI 1146(1cells resulted in prolonged
survival compared to that of nude mice treated with a reporter virus.
These results suggest that Ad-IGF-Ir/as has a therapeutic effect on estab
lished human lung cancer xenografts and may represent an effective and
practical cancer gene therapy strategy.
therapy of a human lung cancer xenograft model.
INTRODUCTION
MATERIALS
The continuous growth of tumors depends on the altered regulation
of the cell cycle. A variety of growth factors and their receptors
mediate the signal transduction pathways that modulate the cell cycle
(1). Among these growth factors are the IGFs1, peptides with molec
Animals, Cells, and Materials. Four-week-old female nude mice were
purchased from Harlan-Sprague-Dawley.
All human lung cancer cell lines
ular weights of about 7,500.000 that can stimulate cellular prolifera
tion and induce cellular differentiation (2). The IGF-Ir is a heterodimer derived from the cleavage of a precursor polypeptide (3),
and recently the roles of IGF-I and the IGF-Ir in cancer cells have
been investigated intensively (4). In certain systems, the IGF-Ir seems
to be essential for malignant transformation because fetal fibroblasts
with a disruption of the IGF-lr gene cannot be transformed by the
SV40 T antigen (5).
The IGF-Ir may also be important for the maintenance of the
malignant state. Trojan et til. (6, 7) demonstrated that antisense IGF-I
could suppress the tumorigenicity of a rat glioblastoma and could
even cause shrinkage of established tumors. Stable transfection of an
antisense plasmid expressing the first 300 bp of the IGF-Ir eliminated
the tumorigenicity of a variety of tumor cell lines and has been
reported to induce systemic effects in established, non-gene-modified
tumors (8, 9). Reduction of IGF-Ir has been shown to induce apoptosis
in tumors, but it produces only growth arrest in untransformed cells
(1). In addition. IGF-receptor knockout mice are viable (although
Received 2/29/96; accepted 4/30/96.
The costs of publication of this article were defrayed in part by the payment of page
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18 U.S.C. Section 1734 solely to indicate this fact.
1 Present address: Department of Internal Medicine. Korea Cancer Center Hospital.
215-4. Gongneung-Dong Nowon-Gu. Seoul 139-240. Korea.
2 To whom requests for reprints should be addressed, at University of Texas South
western Medical Center. 5323 Harry HiñesBoulevard. Dallas. TX 75235-8593. Phone:
(214) 648-4913: Fax: (214) 648-4950.
' The abbreviations used are: IGF. insulin-like growth factor; IGF-Ir. IGF-I receptor;
Ad-IGF-Ir/as. adenovirus expressing an antisense IGF-Ir; Ad-luc. adenovirus expressing
the luciferase gene; m.o.i., multiplicity of infection; CMV, cytomegalovirus.
AND METHODS
(human lung adenocarcinomas A549 and SCC5, human lung large cell carci
noma NCI H460, and human small cell lung carcinoma NCI H82) were
obtained from Adi F. Gazdar (University of Texas Southwestern Medical
Center, Dallas, TX). All cells were passaged in RPMI 1640 with 8% fetal
bovine serum. IGF-I and '^I-labeled IGF-1 were purchased from Amersham.
Construction of Ad-IGF-Ir/as. The cDNA of the IGF-Ir was made by
reverse transcription-PCR of mRNA from NCI H82 (a human small cell lung
cancer cell line). The forward primer used was AGCTG AATTC ATCCC
AAATA AAAGG A, and (he reverse primer used was AGCTG AATTC
GGGGA AGAGG TCTCC GAGGC T. The resulting cDNA fragment con
tained 321 bp of the IGF-Ir cDNA open reading frame, including the ATG
initiation codon. Both ends of the cDNA were engineered to contain EcoRl
restriction sites that were used to clone the fragment in an antisense direction
into the polylinker site of the pAC shuttle plasmid (a gift of Dr. Robert Gerard.
University of Texas Southwestern Medical Center). pAC contains the CMV
immediate early enhancer and promoter and (he SV40 polyadenylation. The
entire insert was sequenced to verify structure, and the resulting pAC-CMVIGF-Ir/as and vector plasmid pJM17 (also a gift of Dr. Gerard) were cotransfected into 293 cells by standard calcium phosphate coprecipitation methods.
Ad-IGF-Ir/as was generated by homologous recombination ( 16). The resulting
adenovirus was confirmed by sequencing of PCR products and plaque purified
three times. A recombinant adenovirus expressing the luciferase gene under
the control of CMV promotor was used as a control virus (Ad-luc).
Cell Growth Assay. Tumor cells were transduced with 10 m.o.i. of AdIGF-Ir/as or Ad-luc for 1 h, and 3 x IO4 cells were plated in 6-well microtiter
plates. These were maintained in complete medium for 48 h and then were
switched to serum-free medium + 0.1% BSA (fraction V) with or without 10
ng/ml of IGF-I. Cell numbers were counted by hemocytometer after 7 days.
Soft Agar Clonogenicity. Anchorage-independent growth was assessed by
soft agar clonogenicity assays. Briefly, tumor cells were transduced with
20 m.o.i. of Ad-IGF-Ir/as and then were detached and plated in 0.2% agarose
with a 1% underlay (5 X 10' cells/plate). After 1 week, medium with 20 m.o.i.
3038
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ANTITUMOR
EFFECTS
OF Ad-IGF-lr/as
2.0
2.0
1.5-
B
1.5 -I
LL
Fig. 1. Scatchard analysis of the IGF-I binding of Adluc-transduced SCC-5 (A) and Ad-IGF-lr/as-transduced
SCC-5 (B).
aS> 1.0-
a
u_ 1.0
CD
i
0.5-
0.5 -
0.0'
234
0.0
Ad lue Bound
of Ad-IGF-lr/as
(Ad-luc as control adenovirus) was added over the soft agar.
The medium overlay was changed after 1 week. Colonies greater than 125 /j.m
were counted after 3 weeks, using a calibrated graticule.
125I-labeled IGF-I Competitive Binding Assay. 125I-labeled IGF-I com
petitive binding assays were performed as described previously (17). Briefly,
human lung cancer cell lines NCI H460 and SCC5 were transduced with
10 m.o.i. of Ad-IGF-lr/as or Ad-luc for 1 h. Cells were then incubated for 24 h
in complete medium, and then the medium was changed to serum-free medium
for another 24 h. Cells were detached with a scraper and were washed with
PBS without Ca+2 and Mg+2. Either 1.0 x IO5 NCI H460 cells or 7 X 10"
SCC5 cells were distributed into tubes and were incubated with 0.5 ml of
RPMI + 0.1% BSA containing varying concentrations of IGF-I and a constant
concentration of 50 pM I25l-labeled IGF-I for 2 h at 4°C.After incubation, cells
were washed twice with RPMI + 0.1% BSA. Cells were then lysed in 0.5 ml
of lysis buffer (0.1% SDS, 0.01 N NaOH. and 0.1% Triton) to measure cell
bound activity. Radioactivity was counted by gamma counter. The IGF-Ir
number was calculated by standard Scatchard analysis (18, 19).
Therapeutic Studies. To assess the effect of Ad-IGF-lr/as on established
tumors, 3.0 x IO5 NCI H460 cells were injected i.p. into 4-week-old nude mice
irradiated with 300 rad. Three days later, 50 m.o.i. (based on the injected tumor
cell number) of Ad-IGF-lr/as was injected i.p. for 5 consecutive days. Controls
consisted of animals injected with PBS and Ad-luc on the same schedule. Mice
were euthanized when they developed preterminal symptoms, and the time to
this point was assessed as survival.
RESULTS
Ad-IGF-lr/as
Reduces the Number of IGF-Irs by U5I-labeled
IGF-I Competitive Binding. We were able to confirm the high level
expression of the IGF-Ir RNA in our human lung cancer cell lines by
RT-PCR using IGF-Ir-specific primers (data not shown). We then
sought to determine whether transduction with the Ad-IGF-lr/as virus
could induce a measurable reduction in the number of surface recep
tors, as measured by a competitive binding assay. In the NCI H460
cells, the observed flmax for control, Ad-luc-, and Ad-IGF-Ir/astransduced cells was 5.10 pM, 6.10 pM, and 3.10 pM, respectively (for
1.0 X 10s cells). The receptor numbers calculated by Scatchard
analysis were 1.53 X 104/cell in the control group, 1.86 X 104/cell in
Ad-luc-transduced group, and 9.3 X 103/cell in the Ad-IGF-Ir/astransduced group. In SCC5 cells, fimax was 7.63 pM in the control
group, 8.23 pM in Ad-luc-transduced group, and 3.75 pM in Ad-IGFIr/as-transduced group (for 7 X IO4 cells; Fig. 1). The calculated
receptor numbers were 3.27 X 104/cell in the control group,
3.52 X 104/cell in the Ad-luc-treated cells, and 1.60 X 104/cell in the
0.5
1.0
1.5
2.0
AdIGF Bound
2.5
3.0
3.5
single bulk transduction, more closely approximating potential clini
cal therapeutic situations than previous studies.
In Vitro Growth Characteristics of Ad-IGF-lr/as-transduced
Human Lung Cancer Cell Lines. The ability of Ad-IGF-lr/as to
block IGF-mediated growth stimulation was tested as described in
"Materials and Methods." In the Ad-luc-transduced group, the addi
tion of IGF-I resulted in an enhancement ratio of 1.44 ±0.18, and in
the Ad-IGF-lr/as-transduced
group, only a 1.15 ±0.08 ratio was
observed (mean ±SE of triplicates). These data suggest that Ad-IGFIr/as-transduction can blunt the mitogenic effect of IGF-I.
Soft Agar Clonogenicity. Previous studies have suggested that the
inhibition of the IGF-Ir has a more profound effect on tumorigenicity
than on in vitro growth on plastic. Therefore, we sought to test the
ability of human lung cancer cells to form colonies in soft agar (Table
1). Three weeks after plating, 448 and 410 colonies were found in
control and Ad-luc-transduced groups, respectively. The Ad-IGF-lr/as
group showed only 68 colonies (an 84% decrease compared to the
Ad-luc group). This finding suggests that Ad-IGF-lr/as is capable of
suppressing the tumorigenicity of NCI H460 cells.
Treatment with Ad-IGF-lr/as Prolongs the Survival of Nude
Mice with Established Human Lung Cancer Xenografts. To test
the therapeutic potential of Ad-IGF-lr/as, nude mice bearing estab
lished i.p. H460 cells were treated 3 days after tumor inoculation as
described in "Materials and Methods." Ad-IGF-lr/as treatment re
sulted in a statistically significant prolongation of survival of these
mice (median survival of 35 days in the control group, 33 days in
Ad-luc-treated group, and 40 days in Ad-IGF-Ir/as-treated group, as
shown in Fig. 2). Thus, there was a 1-week increase in median
survival for animals treated with the antisense virus. No survival
benefit was observed with a once per week for 3 weeks dosing
schedule (data not shown), suggesting that perhaps higher infective
doses would achieve greater therapeutic effect.
DISCUSSION
IGF-I seems to be required for the optimal growth of most normal
cells. Other growth factors vary widely in their necessity in different
cell types (20). The IGF peptides, binding proteins, and receptors have
very important roles in normal growth and development, including
Table 1 Suppression of colony formation
Ad-IGF-Ir/as-treated cells. Therefore, a single transduction by AdIGF-lr/as decreased the receptor number by over 50% compared to the
control virus-transduced cells. It should be stressed that these cells
were not selected in vitro for transduction but were tested after a
3039
in soft agar by transduction
H460Control
Group of NCI
Ad-luciferase-transduced
Ad-IGF-Ir/as-transducedColony
" Each number represents the mean of triplicate plates
of Ad-lGF-lr/as
number"448
410
68
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ANTITUMOR
EFFECTS OF Ad-lGF-Ir/as
25
Fig. 2. Prolongation of survival of i.p. tumor-hearing nude mice by i.p. treatment of
Ad-IGF-Ir/as. Ten animals/group were treated with i.p. injections of virus. Heavy line.
Ad-IGF-Ir/as; dashed line, saline control; thin line. Ad-luc. Log-rank test. P < 0.005.
that of the lung. IGF-mediated growth responsiveness is also found in
most cancer cells. Studies in the past few years have shown that IGFs
are important mitogens in many malignancies and may enhance in
vivo tumor formation, growth, and metastasis (21-23). SV40 T anti
gen, for example, increases the expression of IGF-I, enhancing the
malignant transformation of BALB/c 3T3 cells. Conversely, BALB/c
3T3 cells lacking the IGF-Ir are resistant to malignant transformation
by the SV40 T antigen (6).
The roles of IGF-I and the IGF-Ir have been underscored by a
number of studies indicating the antitumor efficacy of transfection by
antisense constructs. The expression of antisense message to IGF-Ir
renders C6 rat glioblastoma cells nontumorigenic and can cause the
regression of established tumors (7, 8). The transfection of antisense
plasmids to IGF-Ir demonstrates a reduction in tumorigenicity of a
variety of human cancer cell lines, which is at least sometimes
accompanied by the induction of apoptosis (1,9, 24). Surprisingly,
antisense IGF-Ir can cause antitumor effects on preestablished, nongene-modified tumors by unknown (but probably immune-mediated)
mechanisms (24).
The importance of IGFs and the IGF-Ir in human cancers is also
becoming increasingly clear, especially in breast cancer, rhabdomyosarcoma (25), and osteosarcoma (26). Most lung cancer cell
lines express IGF-I and IGF-Ir that mediate autocrine proliferation
(14, 22). In our study, strong expression of IGF-Ir mRNAs were
found in all lung cancer cell lines screened by RT-PCR. Our data
are the first to show that the malignant features of human lung
cancer can also be effectively reduced in vitro and in vivo by even
modest reductions in the level of expression of the IGF-Ir. A
decrease in IGF-Ir expression through the introduction of antisense
constructs may act via the induction of apoptosis in lung cancer
cells as well.
Previous therapeutic strategies designed to interfere with IGFI-mediated signal transduction have used antisense plasmid trans
fection or synthetic antisense oligonucleotides. We are the first to
report the antitumor effects of IGF-Ir inhibition in human lung
cancer cells, and the design and therapeutic effectiveness of an
adenoviral vector expressing antisense IGF-Ir, a potentially more
clinically relevant approach. In our study, a single transduction of
Ad-IGF-Ir/as into human lung cancer cell lines decreased the
IGF-Ir number by 50%. This is comparable to the 70% decrease
observed after stable transfection with an antisense IGF-Ir plasmid
(1, 9). The observed inhibition of growth stimulation by IGF-I in
serum-free medium (from 44% to 14%) is also of a similar mag
nitude to that observed in other systems (24). A 50% decrease in
the receptor number after a single transduction without in vitro
selection indicates that this delivery system is actually very effi
cient, and it is limited by the inherent problems with antisense
approaches. The potential for repeated transduction in a clinical
setting may improve effectiveness.
In spite of the relatively modest effects on the receptor number and
proliferation on plastic, treatment with Ad-IGF-Ir/as induced a dra
matic suppression of colony formation in soft agar, consistent with a
selective effect on the transformed phenotype. We therefore tested the
therapeutic potential of this virus in an i.p. tumor model of the human
lung cancer cell line NCI H460. In this i.p. model, we were able to
observe a statistically significant increased survival. However, com
pared with the treatment effect of antisense plasmid stably transfected
syngeneic rat glioblastoma cells on non-gene-modified tumor cells
(24), this increase in survival is not striking. The systemic antitumor
effects observed for antisense IGF-Ir plasmids in a syngeneic animal
model system are probably the result of an immunity induction in
addition to the reduction of IGF-lr, which would not play a role in this
human tumor xenograft model. In addition, if more effective inhibi
tion of receptor expression could be achieved (perhaps by the use of
dominant negative constructs), the magnitude of the effect may be
improved.
Two important findings were demonstrated in our study: (a) we
demonstrated the in vitro and in vivo antitumor efficacy of antisense
IGF-Ir in human lung cancer cells, which broadens the potential
clinical indications of antisense IGF-Ir therapy in human cancers; and
(b) we demonstrated that the delivery of antisense IGF-Ir via recom
binant adenovirus was effective both in vitro and in vivo. In contrast
to antisense plasmid transfection. adenoviral vectors are a very effi
cient and practical method to deliver therapeutic genes to cancer cells.
The fundamentally nontumor-selective nature of adenovirus transduc
tion is not a concern for this approach because in contrast to its
dramatic effects on tumor cells, antisense IGF-Ir seems to only mar
ginally reduce the growth rate of normal cells, and the expression of
adenoviral vectors is transient, lasting only 1-2 weeks. Thus, Ad-IGFIr/as extends the preclinical data on the inhibition of IGF-Ir to a
potentially practical clinical therapy.
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Antitumor Effects of an Adenovirus Expressing Antisense
Insulin-like Growth Factor I Receptor on Human Lung Cancer
Cell Lines
Choon-Taek Lee, Shan Wu, Dmitry Gabrilovich, et al.
Cancer Res 1996;56:3038-3041.
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