The Efficacy of Small Interfering RNAs Targeted to the Type 1 Insulin-like Growth Factor Receptor (IGF1R) Is Influenced by Secondary
Structure in the IGF1R Transcript*
Erin A.
Bohula
§,
Amanda J.
Salisbury
§,
Muhammad
Sohail§¶,
Martin P.
Playford
,
Johann
Riedemann
,
Edwin M.
Southern¶, and
Valentine M.
Macaulay
From the
Cancer Research UK Laboratories,
Weatherall Institute of Molecular Medicine, Oxford OX3 9DS, United
Kingdom and ¶ Department of Biochemistry, University of Oxford,
Oxford OX1 3QU, United Kingdom
Received for publication, January 22, 2003
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ABSTRACT |
The type 1 insulin-like growth factor receptor
(IGF1R) is often overexpressed by tumors and mediates growth and
apoptosis protection. We previously showed that antisense reagents
complementary to the IGF1R translation start site enhance
radio- and chemosensitivity and impair Atm function. However
these agents induce relatively modest IGF1R down-regulation and affect
insulin receptor levels. To identify alternative sites for molecular
targeting, we utilized scanning oligonucleotide arrays to probe the
secondary structure of IGF1R mRNA. This strategy
enabled selection of antisense oligonucleotides that generated high
heteroduplex yield with IGF1R but not insulin receptor transcripts.
Antisense oligonucleotides that hybridized strongly to
IGF1R mRNA caused IGF1R down-regulation within intact tumor cells, whereas weakly hybridizing oligonucleotides were inactive.
Furthermore, the ability of small interfering RNAs (siRNAs) to block
IGF1R expression correlated with the accessibility of the target
sequence within the transcript. Thus, siRNAs corresponding to weakly
hybridizing oligonucleotides caused minor IGF1R down-regulation, whereas siRNAs homologous to accessible targets induced profound sequence-specific IGF1R gene silencing, blocked IGF
signaling, and enhanced tumor cell radiosensitivity. This indicates
that secondary structure in the target transcript has a major effect on
siRNA efficacy. These findings have implications for siRNA design and
suggest that IGF1R-targeting agents incorporating this mode of action
have potential as anticancer therapy.
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INTRODUCTION |
The type 1 insulin-like growth factor receptor
(IGF1R)1 is often
overexpressed by tumors (1-3), and IGF1R activation mediates tumor
cell proliferation, motility, and protection from apoptosis (4). Tumor
growth can be inhibited in vivo by blocking IGF1R expression
using antisense agents targeting the IGF1R translation start
site (TSS) (5). We previously showed that TSS antisense oligonucleotides (ASOs) and antisense RNA enhanced tumor cell sensitivity to cytotoxic drugs and ionizing radiation and impaired the
function of Atm (6, 7). However we could not suppress IGF1R
expression by more than 80% and sought to identify alternative sites
for molecular targeting.
Intramolecular folding of mRNAs renders all but 5-10% of most
transcripts inaccessible to binding of complementary nucleic acids, but
the complex secondary structure of long mRNAs is not amenable to
accurate modeling (8-11). We used an array-based screen (12) to
identify sites within the human IGF1R transcript that were
accessible to RNase-H-mediated cleavage. This information on secondary
structure allowed us to identify molecular agents that induced IGF1R
down-regulation without affecting insulin receptor (IR) expression.
Structural constraints were shown to govern the activity of ASOs and
also of small interfering RNAs (siRNAs) that mediate RNA interference
(RNAi) in mammalian cells (13).
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MATERIALS AND METHODS |
Oligonucleotides--
A 12-mer deoxyribophosphodiester
oligonucleotide library (dN12) was synthesized as described
(12). Phosphorothioate oligonucleotides were synthesized and
HPLC-purified at the Cancer Research UK Oligonucleotide Laboratory,
South Mimms, UK. RNA/DNA chimeric oligonucleotides were designed as
described (13); the 5' base of each sense strand was immediately
downstream of an AA doublet, and each strand incorporated 19 bases of RNA with two 3' deoxythymidines. An inverted sequence control
duplex was made for each siRNA. These oligonucleotides were synthesized
and HPLC-purified at Transgenomic Laboratories, Glasgow UK.
Complementary strands were annealed in 100 mM potassium acetate, 30 mM Hepes-KOH, pH 7.4, 2 mM
magnesium acetate by incubating at 85 °C for 1 min followed by
37 °C for 1 h. Duplex formation was confirmed by
electrophoresis through 5% low melting temperature agarose (NuSieve
GTG, FMC BioProducts, Rockland ME).
RNase Mapping--
The 1.6-kilobase
HindIII-Asp718 fragment of human IGF1R cDNA
was cloned into HindIII and Asp718-digested pBS
KS- (Stratagene). This plasmid was digested with NotI,
blunt-ended with Klenow enzyme, digested with EcoRV, and
re-ligated to remove extraneous sequence between the T7 promoter and 5'
end of the insert. The resulting construct pIGF1R1581
55 was
linearized with Asp718. End-labeled transcripts representing
1-1581 nt of IGF1R mRNA were made by in
vitro transcription at 30 or 37 °C with 50 µCi of
[
-32P]GTP (>5000 Ci/mmol, Amersham Biosciences), 750 µM each ATP, CTP, and UTP, and 75 µM
unlabeled GTP. Transcripts were purified using MicroSpin G-25 columns
(Amersham Biosciences) and analyzed on 6% denaturing polyacrylamide
gels. For RNase mapping reactions, 5 fmol of 5' end-labeled transcript
were incubated with 0.5 units of RNase H and 0-500 pmol of
deoxyribophosphodiester oligonucleotide library (dN12), and
the products were analyzed by gel electrophoresis.
Antisense Oligonucleotide Scanning Arrays--
Arrays were made
as described (12) using amidated polypropylene (Beckman Instruments,
Fullerton, CA) as substrate. The synthesis was carried out using
standard nucleotide-cyanoethyl-phosphoramidites on an adapted
ABI 394 DNA synthesizer (Applied Biosystems). Reagents were delivered
to the substrate surface using a diamond-shaped mask with a 30-mm
diagonal. The mask was sealed against the polypropylene to create a
cell into which reagents for first base synthesis were delivered and
then removed. After each nucleotide coupling the mask was displaced by
1.5-mm steps to create arrays of 1-20-mer antisense oligonucleotides.
Internally radiolabeled IGF1R1581 transcripts were obtained
by in vitro transcription with T7 RNA polymerase, with 20 µCi of [
-32P]UTP (~3000 Ci/mmol, Amersham
Biosciences), 750 µM each ATP, CTP, and GTP, and 18.75 µM unlabeled UTP. A 2.5-kilobase
SalI-PstI fragment of human IR cDNA was
cloned into pBluescript SK and linearized with PstI, and
internally radiolabeled transcripts representing bases 1-2499 were
made as above. Array hybridizations were performed in 1 M
NaCl, 10 mM Tris-Cl, pH 7.4, 1 mM EDTA, 0.01%
SDS at 25 or 37 °C using 50-60 fmol of transcript. Arrays were
washed and imaged using the program xvseq as described (14).
RNase H Assays for Analysis of Individual
Oligonucleotides--
End-labeled IGF1R transcripts were
obtained by in vitro transcription at 37 °C (as above)
and purified through Mini Quick Spin RNA Columns (Roche Molecular
Biochemicals). RNase H assays used 0.5 fmol of end-labeled transcript
incubated in 50 mM Tris-Cl, pH 7.4, 50 mM KCl,
10 mM MgCl2, 1 mM dithiothreitol, 1 units of RNase H (Promega), 20 units of RNase inhibitor (Promega) with 0.5-50 fmol of ASO in 10 µl. After 30 min at 37 °C reactions were terminated by the addition of 10 µl of formamide gel loading buffer, and the products were analyzed by electrophoresis through 6%
denaturing polyacrylamide gels.
Cell Culture and Transfection--
Tumor cell lines MDA-MB-231
(human estrogen receptor negative breast cancer), ME (human
melanoma (15), A549 (human non-small cell lung cancer), DU145 (human
androgen-resistant prostate cancer), UC101 (human ovarian cancer), and
B16.F1 (mouse melanoma) were cultured in RPMI 1640 medium with 10%
fetal calf serum, and all were negative for mycoplasma infection. Cells
were transfected with ASOs using Cytofectin (Glen GSV) or
Oligofectamine (Invitrogen) according to the manufacturer's
instructions. All siRNA transfections were performed using
Oligofectamine. Total RNA was made using RNeasy columns (Qiagen) and
0.5 µg of each sample was analyzed for expression of IGF1R and actin
using the Access reverse transcription-PCR kit (Promega) according to
the manufacturer's instructions. The sequences of PCR primers were:
IGF1R forward, 5'-gaaatctgcgggccaggcatcg-3'; IGF1R reverse,
5'-ctccatggtccctggacacaggt-3'; actin forward,
5'-tcatgaagtgtgacgttgacatccgt-3'; actin reverse,
5'-cctagaagcatttgcgttgcacgatg-3'. PCR cycles were 48 °C for 45 min,
94 °C for 2 min, followed by 20 cycles (shown in preliminary
reactions to give quantitative yield of product) of 94 °C for
30 s, 54 °C for 45 s, 68 °C for 2 min, with a final 7 min at 68 °C. After 48 h some transfected cultures were
serum-starved overnight and stimulated with 50 nM
long-R3-IGF-I (GroPep). Cells were lysed and analyzed as described (6)
by immunoblotting for IGF1R (Santa-Cruz), IR (Santa Cruz),
phospho-Ser-473 Akt (Cell Signaling Technology), total Akt (Cell
Signaling Technology), and
-tubulin (Sigma). Immunofluorescent
detection of the IGF1R used antibody
IR3 (Ab1, Oncogene Science) as
described (16). Activated IGF1R was detected by immunoprecipitation for
phosphotyrosine (P-Tyr-100, Cell Signaling Technology) and
immunoblotting for IGF1R
-subunit. To measure survival, cells were
disaggregated 24 h after transfection and re-seeded in 6-cm dishes
at 2000 cells/dish. After 4-6 h to allow adherence, some dishes were
irradiated in a 137Cs source at 3 gray/min. After
1-2 weeks of incubation at 37 °C, visible colonies were stained and
counted (7). Statistical analysis was performed with GraphPad Prism®
3.0c software using the paired t test for comparisons of two
treatments and analysis of variance for multiple comparisons.
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RESULTS |
We used a two-step empirical screen (12, 14) to investigate
binding accessibility in the IGF1R transcript and to
identify structurally homologous regions within the insulin receptor
transcript. First, RNase mapping identified broad regions of
accessibility within the 5' 1.6 kilobases of the IGF1R
transcript (Fig. 1A). Based on
the relative yield of cleavage products and the mixed nature of the
sequence, we selected for further study a 150-nt region corresponding
to bases 536-685 of the human IGF1R sequence (17). To
locate more precisely the regions of high and low accessibility, we
synthesized ASO-scanning arrays complementary to this region. The ASOs
were covalently bound by their 3' ends to the aminated polypropylene
substrate and represented all possible complementary sequences of 1-20
residues within the selected region. Probing with IGF1R
mRNA at 37 °C (Fig. 1B) showed negligible binding to monomers at the edges of the array. Along the center line 20-mers showed significant heteroduplex yield only in the accessible regions of
transcript, with three peaks of hybridization in the region 590-668
nucleotides (peaks 1, 2, 4; Fig.
1B). Hybridizations at 25 °C resulted in higher
heteroduplex yield (not shown), with a similar hybridization pattern
and an additional focus of hybridization not present at 37 °C
(region 3, Fig. 1B). We also hybridized the array
to human IR mRNA to identify ASOs that could affect
expression of the IR, which has 60% sequence homology with the IGF1R
(Fig. 1B). This analysis revealed two peaks of heteroduplex
formation between IR mRNA and the IGF1R ASOs on the
array (lower panel, Fig. 1B).

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Fig. 1.
Identification of sites within IGF1R
or IR mRNA accessible to hybridize with IGF1R
ASOs. A, RNase mapping. IGF1R mRNA (5 fmol) was incubated with RNase H in the absence or presence of 5, 10, 20, 50, 100, or 500 pmol of random 12-mers (dN12).
Asterisk, region selected for array screen. B,
hybridization of IGF1R or IR mRNA to scanning
array of IGF1R ASOs. Middle panel, hybridization to
IGF1R transcript. The histogram (upper panel)
represents quantification of binding of 20-mer ASOs to IGF1R
mRNA. Lower panel, hybridization to IR
transcript. Numbered arrowheads, ASO sequences selected for
further study. The 5' end of the sense sequence (17) targeted by ASO1
was base 596, that targeted by ASO2 was base 612, that targeted by ASO3
was base 622, that targeted by ASO4 was base 637, and that targeted by
ASO6 was base 546. C, left, 5' end-labeled
IGF1R transcript was incubated with RNase H in the absence
of ASO or with ASOs 1, 2, 6 or TSS ASO at increasing molar excesses of
ASO:transcript (1:1, 5:1, 20:1, 30:1, 100:1). Arrowheads,
full-length ~1600-nt transcript, 600-nt cleavage product of ASOs 1 and 2, 50-nt product of TSS ASO. Asterisk, nonspecific
product present in all lanes. The lower part of the gel is shown at
longer exposure to visualize the 50-nt product. Right,
analysis (mean ± S.E.) of 3 assays, expressed as % 600-nt
cleavage product. A greater yield of product is shown in the presence
of 20:1 to 100:1 ASOs 1 and 2 than ASO6, and ASO2 was more effective
than ASO1 at 5:1 and 20:1 (p < 0.05 for each
comparison).
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We synthesized 20-mer phosphorothioate ASOs that showed intense (ASOs
1, 2, 4) or negligible (ASOs 3, 6) binding to IGF1R mRNA
at 37 °C (Fig. 1B and Table
I). In a cell-free assay, these ASOs
induced RNase H-mediated cleavage of IGF1R mRNA that was proportional to the relative affinity of hybridization when tethered on
the scanning array (Fig. 1C). Effects on IGF1R expression in human tumor cells were assessed using transfection conditions that
resulted in uptake of fluorescently tagged ASO into 90-95% of cells
5-24 h after transfection (not shown). We observed significant IGF1R
down-regulation in MDA-MB-231 breast cancer cells transfected with 300 nM ASOs 1 (p < 0.01), 2 (p < 0.01), and 4 (p < 0.05) that hybridized strongly to
the transcript at 37 °C (Fig. 2,
A and B). There was no effect on IGF1R expression
in cells transfected with ASOs 3 and 6 that hybridized weakly at
37 °C (Fig. 2, A and B). The same patterns of
relative efficacy were observed in ME human melanoma and B16.F1 murine
melanoma (not shown).
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Table I
Sequences of oligonucleotides used in this study
The table shows sequences of ASOs and siRNAs. Phosphorothioate ASOs
were used for RNase H assays and for cell transfection. All siRNAs were
21-mer chimeric RNA/DNA duplexes, with the exception of 18-27-mer
variants of R2. The sequence of duplex Mut2 was as for R2 with the
exception of a single bp mutation
(underlined).
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Fig. 2.
Antisense oligonucleotides that hybridize to
IGF1R mRNA in vitro cause
sequence-specific inhibition of IGF1R expression. A,
immunoblots for IGF1R of MDA-MB-231 cell lysates after transfection
with 30-300 nM ASOs 1, 3, or scrambled sequence
(Scr) controls. B, graph shows the mean ± S.E. of 3-5 independent evaluations of each ASO transfected at 30 or
300 nM. IGF1R levels were corrected for loading differences
and expressed as % levels in cells treated with equivalent
concentrations of scrambled oligonucleotide. C, MDA-MB-231
breast cancer cells were transfected with 30-300 nM ASO4,
TSS ASO, or scrambled controls and immunoblotted for IGF1R and
IR.
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We compared the novel ASOs with an IGF1R ASO complementary to the IGF1R
TSS, the conventional site for antisense targeting (5). In cell-free
RNase H assays, this sequence generated an indistinct 50-nt cleavage
product (Fig. 1C). In transfected MDA-MB-231 cells ASOs 1, 2, and 4 caused significantly greater IGF1R down-regulation than the
TSS ASO at 30 nM (p < 0.01 for each
comparison), but there was no difference at 300 nM (Fig.
2B). In addition we saw evidence of modest but consistent
down-regulation of the insulin receptor by 300 nM TSS ASO
(69 ± 8% of levels in scrambled control-treated cells compared
with 112 ± 11% for ASO4, p < 0.01; Fig.
2C). We did not investigate secondary structure in the
region of the IGF1R translation start site, and the TSS ASO has
negligible homology to the start site of the IR transcript.
However several regions of IR mRNA have 4-5 bases of
homology with the TSS ASO, which may be sufficient (if accessible to
binding) to induce RNase H activity (18). The array screen had revealed
two regions of heteroduplex formation between IR mRNA
and the IGF1R ASO array, one immediately upstream of ASO1 (Fig.
1B). This ASO hybridized with ~30-fold greater intensity
to the IGF1R transcript than to the IR and caused
minimal effects on IR expression (85-90% of control levels at
150-300 nM). There was no detectable IR down-regulation in
cells transfected with ASOs 2 or 4 (Fig. 2C and data not
shown). Thus, the scanning array permitted identification of ASOs that induced sequence-specific IGF1R down-regulation without affecting expression of the IR.
This study was extended to evaluate the effects of secondary structure
on IGF1R down-regulation induced by siRNAs. We synthesized a 21-mer RNA
duplex R2 corresponding to ASO2, which was immediately downstream of
the required AA motif (13), and that was the most intensely
hybridizing ASO from the scanning array (Fig. 1B). Duplex R2
induced profound sequence-specific IGF1R gene silencing, to ~1% of levels in cells treated with an inverted control duplex (Fig.
3). This suggests that the inability to
suppress IGF1R expression by greater than 80% using antisense was
unlikely to be due to poor transfection efficiency but was more likely
a fundamental limitation of the AS approach. An 18-mer R2 duplex with a
3-bp 3' deletion was less effective than the 21-bp R2, whereas
comparable IGF1R down-regulation was induced by 24- and 27-mer duplexes
representing 3- and 6-bp 3' extensions of R2 (see Table I and Fig.
3D). This is in contrast to duplex length requirements for
RNAi in Drosophila (19) but consistent with recently
reported characteristics of RNAi in mammalian cytoplasmic lysate
(20).

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Fig. 3.
Profound IGF1R gene silencing
induced by siRNA equivalent to IGF1R ASO that hybridizes intensely to
IGF1R mRNA. A, human ME melanoma cells
were transfected with Oligofectamine alone (Nil) or with 200 nM TSS scrambled sequence control (Scr) or TSS
ASO or with siRNA R2 or inverted sequence duplex (Inv2).
Cells were lysed 48 h after transfection and analyzed by
immunoblotting for IGF1R. Arrow, ~220-kDa IGF1R
proreceptor. B, sequence specificity of IGF1R
gene silencing in ME melanoma cells transfected with Oligofectamine
alone or with 5, 50, or 500 nM inverted sequence duplex or
R2. Cell lysates were analyzed by immunoblotting for IGF1R and IR.
Similar results were obtained in a second set of independently prepared
ME cell lysates, in MDA-MB-231 breast cancer and UC101 ovarian cancer
cells. C, immunofluorescent staining of MDA-MB-231 cells
48 h after transfection with 100 nM inverted sequence
duplex or R2. Red, IGF1R; blue, Hoescht stain for
DNA. D, MDA-MB-231 cells were transfected with 0.5 or 5 nM R2 or with 18-27-mer variants of the same duplex (see
Table I). IGF1R protein levels were analyzed after 48 h.
Arrow, ~220-kDa IGF1R proreceptor.
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The effects of R2 were compared at the RNA and protein levels with R6,
a duplex corresponding to ASO6 that failed to hybridize to
IGF1R mRNA on the array (Fig. 1B). Compared
with R6, duplex R2 caused greater inhibition of expression of the
IGF1R measured by reverse transcription-PCR (Fig.
4A) and induced more profound dose-dependent reduction in IGF1R protein levels than R6
(p < 0.05 at 0.5 and 50 nM,
p < 0.01 at 5 nM; Fig. 4, B and
C). Indeed the difference between these two duplexes was
greater than that between R2 and a mutant R2 duplex (Fig. 4,
B and C). It was notable that in this system a
single base pair mutation reduced but did not abolish siRNA activity.
R2 and R6 were also compared in a range of human and murine cell lines,
and in all cases R2 caused significantly more profound IGF1R
down-regulation (DU145 (p < 0.01), A549
(p < 0.05), ME (p < 0.01), B16
(p < 0.05); Fig. 4D). This indicates that
secondary structure in the transcript has a major effect on siRNA
efficacy and suggests that the structural features dictating access are
robust and conserved between different cell lines and species.

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Fig. 4.
Influence of mRNA secondary structure on
siRNA efficacy. A, reverse transcription-PCR analysis
of IGF1R expression in UC101 ovarian cancer cells
transfected with 100 nM siRNAs. Upper
panel, 473-bp IGF1R product. Lower panel, 287-bp actin
product. The same result was obtained in a second set of independently
prepared RNAs. B, immunoblot for IGF1R in MDA-MB-231 cells
48 h after transfection with R2, Mut2 (sequence as R2 with a
single-bp mutation in the center of the duplex), or R6 at 0.5, 5, or 50 nM. C, analysis of IGF1R protein levels in
siRNA-transfected MDA-MB-231 cells. Points represent the mean (± the
range of values from two independent experiments) of IGF1R levels,
expressed as % value in cells treated with equivalent concentrations
of inverted control duplex. D, analysis of IGF1R levels
(mean ± S.E. of triplicate experiments) in human DU145 prostate
cancer, A549 non-small cell lung cancer, ME melanoma, and murine B16.F1
melanoma cells transfected with 10 nM R2 or R6.
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We then assessed the efficacy of a second pair of duplexes based on the
sequence around peak 4 of hybridization between IGF1R mRNA and the IGF1R ASO scanning array (Fig.
5A). Duplexes R4 and R5 were
designed to target 19-mer sequences immediately downstream of AA
motifs at bases 636 and 639, respectively, of the IGF1R sequence (17).
Data from the scanning array indicated that there was a ~6.5-fold
difference in hybridization intensity between the equivalent 19mer ASOs
(Fig. 5B). Both duplexes induced IGF1R down-regulation in
human ovarian and prostate cancer cells, but R4 was significantly more
potent (p < 0.05) in both cell lines (Fig. 5,
C and D). Thus, a 3-bp shift had major effects on
siRNA efficacy, paralleling differences in heteroduplex yield on the scanning array.

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Fig. 5.
Small sequence shift has major effect on
hybridization intensity and siRNA efficacy. A,
phosphorimaging analysis of the peak 4 region of hybridization of
IGF1R mRNA to 19-mer IGF1R ASOs on the scanning array.
ASOs equivalent to duplexes R4 and R5 are marked by
arrowheads and by black bars in the histogram.
B, the table shows hybridization intensity (arbitrary units,
from the scanning array) of 19-mer ASOs equivalent to duplexes R2, R4,
R5, and R6. C, representative immunoblot showing effects of
10 nM siRNAs or inverted controls (Inv2) on
IGF1R levels in human DU145 prostate cancer cells. D,
results (mean ± S.E.) of triplicate analyses of IGF1R protein
levels in DU145 prostate cancer and UC101 ovarian cancer cells
transfected with 10 nM R4 or R5.
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Next we investigated the effects of ASOs and siRNA on intracellular
signaling. The relatively modest IGF1R down-regulation induced by ASOs
1-6 or siRNA R6 was not sufficient to abolish IGF-I signaling to Akt
(Fig. 6A and data not shown).
In contrast transfection with R2 induced profound IGF1R gene
silencing such that we could not detect IGF-I-induced phosphorylation
of the IGF1R
-subunit or of Akt, a key intermediate in the major
anti-apoptosis pathway downstream of the IGF1R (4). Finally we compared
the effects on colony-forming efficiency and post-irradiation survival of transfection with 100 nM ASO4 or siRNA duplexes R4 and
R5, equivalent to strongly and weakly hybridizing ASOs, respectively. The phosphorothioate ASO4 caused inhibition of overall survival and
survival after 2 gray irradiation (SF2), but this effect was entirely
nonspecific, with similar results in cultures treated with the
scrambled sequence oligonucleotide (Scr4, Fig. 6, B and C). Transfection with siRNAs R4 or R5 inhibited survival of
MDA-MB-231 (p < 0.001 for comparison of each siRNA
with inverted control duplex), with a significantly greater effect in
R4-treated cells (p < 0.01 for comparison with R5;
Fig. 6B). Only in cultures treated with the more potent
siRNA duplex R4 was there significant inhibition of survival after 2 gray irradiation (SF2; p < 0.01 for comparisons with
Inv4 and R5; Fig. 6C). These results confirm that the
complex secondary structure of target transcripts can influence siRNA efficacy and that these differences are sufficient to confer major variation in biological efficacy of potential therapeutic
significance.

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Fig. 6.
siRNAs corresponding to intensely hybridizing
ASOs block IGF signaling and post-irradiation survival.
A, MDA-MB-231 cells transfected with 100 nM RNA
duplexes were serum-starved overnight and treated with 50 nM IGF-I for 30 min. Comparable results were seen in two
sets of independently prepared MDA-MB-231 cell lysates. PY,
phosphotyrosine; IP, immunoprecipitation; IB,
immunoblot. B, clonogenic survival in MDA-MB-231
cells after transfection with 100 nM siRNA, inverted
control duplexes (Inv), ASO4, or scrambled control
(Scr4). Results represent the mean ± S.E. of colony
counts in triplicate dishes. Similar results were obtained in two
further clonogenic assays. C, clonogenic survival of
transfected MDA-MB-231 cells that were unirradiated or irradiated at 2 gray. The fraction surviving 2 gray irradiation (SF2) is shown as the
mean ± S.E. of three independent assays.
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DISCUSSION |
We previously showed correlation between array hybridization and
effects on gene expression, by assessing in vitro
translation of cyclin B transcripts following ASO microinjection into
Xenopus oocytes (12). Here, we have shown that intensity of
array hybridization at 37 °C successfully predicted for target
down-regulation within intact tumor cells. This is despite the fact
that array conditions were non-physiological; ASOs were tethered at the
3' ends, and the composition of the hybridization buffer did not
reflect the protein-rich cytosolic microenvironment. Furthermore,
hybridizations were performed in 1 M NaCl since there was
insignificant heteroduplex formation at physiological (150 mM) salt concentration (not shown). In a search for
effective IGF1R ASOs to combat epithelial hyperplasia in psoriasis,
Wraight et al. (21) used a program designed to predict
regions of the transcript lacking internal duplex and hairpin structure
(21). One of the selected 15-mer ASOs fell within the region we
screened, targeting nt 653-667 of the IGF1R transcript.
This was found to be ineffective and indeed was shown to hybridize
weakly on our scanning array (relative hybridization intensity ~0.03
compared with 1.0 for ASO2; Fig. 1B). In accordance with our
results, even the most effective ASOs suppressed IGF1R expression only
to ~25% of control levels (21).
We found major differences in the activity of IGF1R siRNAs, paralleled
by changes in the intensity of hybridization of the equivalent ASOs to
IGF1R mRNA. There have been two recent reports of
differential efficacy of siRNAs, against human immunodeficiency virus-1
rev (22) and Tissue Factor (23), attributed in the latter report to
protein binding at/near the siRNA target site. This is an unlikely
explanation for our findings since we observed parallel differences in
ASO and siRNA efficacy within intact cells and in cell-free (and
virtually protein-free) RNase H assays. The parallels we observed
between array hybridization and siRNA efficacy suggest that secondary
structure in the IGF1R transcript was a major factor in
determining the efficacy of synthetic 21-mer siRNAs. Unlike
phosphorothioate ASOs that induce RNase H activity, siRNAs act as
"guide sequences" to direct the RNA-induced silencing complex to
the homologous sequence in the target transcript (24). The precise
nature of the molecular interaction between siRNAs and target mRNA
is unclear. The need for access comparable with that required for ASO
binding supports the concept, as originally proposed when RNAi was
first recognized (24, 25), of direct interaction by base-pairing
between the transcript and component(s) of the duplex. This is
consistent with the recent demonstration that antisense strands can
mediate RNAi in mammalian cytoplasmic lysate (20).
The success rate for effective siRNA design is clearly higher than the
~10% reported for ASOs (10, 26). However a requirement for access
could explain why some siRNAs are ineffective. Elbashir et
al. (13) were unable to down-regulate vimentin in HeLa cells, but
in a later report vimentin expression was effectively silenced by
siRNAs directed against different regions of the same transcript (26).
We have demonstrated here that mRNA folding has major consequences
for siRNA-induced gene silencing, with clear implications for the
design of effective siRNAs. Furthermore, the potency of these siRNAs in
blocking IGF signaling and tumor cell survival suggests that
IGF1R-targeting agents incorporating this mode of action have potential
as anticancer therapy.
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ACKNOWLEDGEMENTS |
We are grateful to Dr. Renato Baserga, Kimmel
Cancer Centre, Thomas Jefferson University, Philadelphia for human
IGF1R cDNA and to Dr. Joe Barr, Howard Hughes Medical Institute
Research Laboratories, University of Chicago, Illinois for human
insulin receptor cDNA.
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Note Added in Proof |
After this manuscript was submitted,
Vickers et al. (27) reported a correlation between
transcript sites effectively targeted by siRNAs and by optimized ASOs,
supporting the conclusion that siRNA efficacy is affected by mRNA
secondary structure.
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FOOTNOTES |
*
This work was supported by Cancer Research UK, the
Medical Research Council, the Rhodes Trust, and the Royal College of
Physicians.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Joint first authors.
To whom correspondence should be addressed: Cancer Research UK
Molecular Oncology Laboratories, Weatherall Institute of Molecular Medicine, Headley Way, Headington, Oxford OX3 9DS, UK. Tel.:
44-1865-222433; Fax: 44-1865-222431; E-mail:
macaulay@cancer.org.uk.
Published, JBC Papers in Press, February 24, 2003, DOI 10.1074/jbc.M300714200
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ABBREVIATIONS |
The abbreviations used are:
IGF1R, type 1 insulin-like growth factor receptor;
ASO, antisense oligonucleotide;
IR, insulin receptor;
siRNA, small interfering RNA;
TSS, translation
start site: HPLC, high performance liquid chromatography;
RNAi, RNA
interference;
nt, nucleotide(s);
Atm, ataxia telangiectasia-mutated;
AA, adenosine doublet.
 |
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