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

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 toIGF1R 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. 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 toIGF1R 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. type 1 insulin-like growth factor receptor antisense oligonucleotide insulin receptor small interfering RNA translation start site: HPLC, high performance liquid chromatography RNA interference nucleotide(s) ataxia telangiectasia-mutated adenosine doublet The type 1 insulin-like growth factor receptor (IGF1R)1 is often overexpressed by tumors (1Hellawell G.O. Turner G.D. Davies D.R. Poulsom R. Brewster S.F. Macaulay V.M. Cancer Res. 2002; 62: 2942-2950PubMed Google Scholar, 2Kanter-Lewensohn L. Dricu A. Girnita L. Wejde J. Larsson O. Growth Factors. 2000; 17: 193-202Crossref PubMed Scopus (60) Google Scholar, 3Hakam A. Yeatman T.J. Lu L. Mora L. Marcet G. Nicosia S.V. Karl R.C. Coppola D. Hum. Pathol. 1999; 30: 1128-1133Crossref PubMed Scopus (216) Google Scholar), and IGF1R activation mediates tumor cell proliferation, motility, and protection from apoptosis (4Baserga R. Hongo A. Rubini M. Prisco M. Valentinis B. Biochim. Biophys. Acta. 1997; 1332: 105-126Crossref PubMed Scopus (480) Google Scholar). Tumor growth can be inhibited in vivo by blocking IGF1R expression using antisense agents targeting the IGF1R translation start site (TSS) (5Resnicoff M. Coppola D. Sell C. Rubin R. Ferrone S. Baserga R. Cancer Res. 1994; 54: 4848-4850PubMed Google Scholar). 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 (6Macaulay V.M. Salisbury A.J. Bohula E.A. Playford M.P. Smorodinsky N.I. Shiloh Y. Oncogene. 2001; 20: 4029-4040Crossref PubMed Scopus (115) Google Scholar, 7Hellawell G.O. Ferguson D.J.P. Brewster S.F. Macaulay V.M. BJU Int. 2003; 91: 271-277Crossref PubMed Scopus (62) Google Scholar). 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 (8Lima W.F. Monia B.P. Ecker D.J. Freier S.M. Biochemistry. 1992; 31: 12055-12061Crossref PubMed Scopus (214) Google Scholar, 9Michel F. Westhof E. J. Mol. Biol. 1990; 216: 585-610Crossref PubMed Scopus (1106) Google Scholar, 10Stein C.A. J. Clin. Invest. 2001; 108: 641-644Crossref PubMed Scopus (157) Google Scholar, 11Sohail M. Akhtar S. Southern E.M. RNA (N. Y.). 1999; 5: 646-655Crossref PubMed Scopus (66) Google Scholar). We used an array-based screen (12Sohail M. Hochegger H. Klotzbucher A. Guellec R.L. Hunt T. Southern E.M. Nucleic Acids Res. 2001; 29: 2041-2051Crossref PubMed Scopus (57) Google Scholar) 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 (13Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8018) Google Scholar). A 12-mer deoxyribophosphodiester oligonucleotide library (dN12) was synthesized as described (12Sohail M. Hochegger H. Klotzbucher A. Guellec R.L. Hunt T. Southern E.M. Nucleic Acids Res. 2001; 29: 2041-2051Crossref PubMed Scopus (57) Google Scholar). 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 (13Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8018) Google Scholar); 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 mmmagnesium 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). The 1.6-kilobaseHindIII-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 Δ55 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 [γ-32P]GTP (>5000 Ci/mmol, Amersham Biosciences), 750 μm each ATP, CTP, and UTP, and 75 μmunlabeled 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. Arrays were made as described (12Sohail M. Hochegger H. Klotzbucher A. Guellec R.L. Hunt T. Southern E.M. Nucleic Acids Res. 2001; 29: 2041-2051Crossref PubMed Scopus (57) Google Scholar) 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 [α-32P]UTP (∼3000 Ci/mmol, Amersham Biosciences), 750 μm each ATP, CTP, and GTP, and 18.75 μm unlabeled UTP. A 2.5-kilobaseSalI-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 mNaCl, 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 (14Sohail M. Southern E.M. Adv. Biochem. Eng. Biotechnol. 2002; 77: 43-56PubMed Google Scholar). 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. Tumor cell lines MDA-MB-231 (human estrogen receptor negative breast cancer), ME (human melanoma (15Dunbar P.R. Smith C.L. Chao D. Salio M. Shepherd D. Mirza F. Lipp M. Lanzavecchia A. Sallusto F. Evans A. Russell-Jones R. Harris A.L. Cerundolo V. J. Immunol. 2000; 165: 6644-6652Crossref PubMed Scopus (125) Google Scholar), 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 nmlong-R3-IGF-I (GroPep). Cells were lysed and analyzed as described (6Macaulay V.M. Salisbury A.J. Bohula E.A. Playford M.P. Smorodinsky N.I. Shiloh Y. Oncogene. 2001; 20: 4029-4040Crossref PubMed Scopus (115) Google Scholar) by immunoblotting for IGF1R (Santa-Cruz), IR (Santa Cruz), phospho-Ser-473 Akt (Cell Signaling Technology), total Akt (Cell Signaling Technology), and β-tubulin (Sigma). Immunofluorescent detection of the IGF1R used antibody αIR3 (Ab1, Oncogene Science) as described (16Ligensa T. Krauss S. Demuth D. Schumacher R. Camonis J. Jaques G. Weidner K.M. J. Biol. Chem. 2001; 276: 33419-33427Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Activated IGF1R was detected by immunoprecipitation for phosphotyrosine (P-Tyr-100, Cell Signaling Technology) and immunoblotting for IGF1R β-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 (7Hellawell G.O. Ferguson D.J.P. Brewster S.F. Macaulay V.M. BJU Int. 2003; 91: 271-277Crossref PubMed Scopus (62) Google Scholar). 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. We used a two-step empirical screen (12Sohail M. Hochegger H. Klotzbucher A. Guellec R.L. Hunt T. Southern E.M. Nucleic Acids Res. 2001; 29: 2041-2051Crossref PubMed Scopus (57) Google Scholar, 14Sohail M. Southern E.M. Adv. Biochem. Eng. Biotechnol. 2002; 77: 43-56PubMed Google Scholar) 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 IGF1Rtranscript (Fig. 1 A). 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 (17Ullrich A. Gray A. Tam A.W. Yang-Feng T. Tsubokawa M. Collins C. Henzel W. T. S. E. S. J. Y. J. 5: PubMed Scopus Google Scholar). To more the regions of high and low we synthesized arrays complementary to this The ASOs were by 3′ to the polypropylene substrate and all complementary sequences of within the selected with at 37 °C (Fig. 1 showed binding to at the of the the showed heteroduplex yield in the accessible regions of with of in the region at 25 °C in heteroduplex yield with a and an of not at 37 °C 1 We also hybridized the to human IR mRNA to identify ASOs that could affect expression of the which has sequence with the IGF1R (Fig. 1 This analysis two of heteroduplex formation between IR mRNA and the IGF1R ASOs on the 1 We synthesized ASOs that showed or binding to IGF1R mRNA at 37 °C (Fig. 1 and a these ASOs induced RNase cleavage of IGF1R mRNA that was to the relative of on the scanning (Fig. 1 on IGF1R expression in human tumor cells were using transfection that in of ASO into of cells h after transfection We IGF1R down-regulation in MDA-MB-231 breast cells transfected with ASOs 1 2 and that hybridized strongly to the transcript at 37 °C (Fig. and was effect on IGF1R expression in cells transfected with ASOs 3 and that hybridized weakly at 37 °C (Fig. A and The of relative were in ME human melanoma and B16.F1 melanoma of oligonucleotides used in this sequences of ASOs and Phosphorothioate ASOs were used for RNase H assays and for cell All siRNAs were chimeric RNA/DNA with the of of The sequence of duplex was as for with the of a in a oligonucleotides that to IGF1R mRNA in vitro sequence-specific of IGF1R expression. for IGF1R of MDA-MB-231 cell after transfection with ASOs or sequence the of of each ASO transfected at 30 or IGF1R were for loading and as in cells with of MDA-MB-231 breast cells were transfected with TSS or and for IGF1R and The sequences of ASOs and Phosphorothioate ASOs were used for RNase H assays and for cell All siRNAs were chimeric RNA/DNA with the of of The sequence of duplex was as for with the of a We the ASOs with an IGF1R ASO complementary to the IGF1R the site for antisense targeting (5Resnicoff M. Coppola D. Sell C. Rubin R. Ferrone S. Baserga R. Cancer Res. 1994; 54: 4848-4850PubMed Google Scholar). RNase H this sequence generated an cleavage (Fig. 1 transfected MDA-MB-231 cells ASOs and caused IGF1R down-regulation than the TSS ASO at 30 for each but was at addition we of modest but down-regulation of the insulin receptor by TSS ASO of in cells with for We not investigate secondary structure in the region of the IGF1R translation start and the TSS ASO has to the start site of the IR transcript. However regions of IR mRNA have bases of with the TSS which be accessible to to induce RNase H activity H. Nucleic Acids Res. PubMed Scopus Google Scholar). The screen two regions of heteroduplex formation between IR mRNA and the IGF1R ASO immediately of This ASO hybridized with to the IGF1R transcript than to the IR and caused on IR expression of control at was IR down-regulation in cells transfected with ASOs 2 or (Fig. 2 and not Thus, the scanning of ASOs that induced sequence-specific IGF1R down-regulation without affecting expression of the This study was to the of secondary structure on IGF1R down-regulation induced by We synthesized a RNA duplex corresponding to which was immediately downstream of the AA (13Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8018) Google Scholar), and that was the most hybridizing ASO from the scanning (Fig. 1 Duplex induced profound sequence-specific IGF1R gene silencing, to of in cells with an inverted control duplex This that the to suppress IGF1R expression by than 80% using antisense was to be to transfection but was more a of the An duplex with a 3′ was than the whereas IGF1R down-regulation was induced by and representing and 3′ of and This is in to duplex for in S.M. J. A. Lendeckel W. Tuschl T. J. 2001; 20: PubMed Scopus Google Scholar) but with of in mammalian J. A. H. R. Tuschl T. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The of were at the RNA and with a duplex corresponding to that to toIGF1R mRNA on the (Fig. 1 with duplex caused of expression of by reverse transcription-PCR and induced more profound in IGF1R than at 0.5 and 50 at 5 the between these two was than that between and a duplex (Fig. and was that in this a base but not siRNA and were also in a of human and cell and in all caused more profound IGF1R down-regulation A549 ME This indicates that secondary structure in the transcript has a major effect on siRNA and that the and between cell lines and We then the of a of on the sequence of between and the IGF1R ASO scanning A). and were designed to target sequences immediately downstream of AA at bases and of the IGF1R sequence (17Ullrich A. Gray A. Tam A.W. Yang-Feng T. Tsubokawa M. Collins C. Henzel W. T. S. E. S. J. Y. J. 5: PubMed Scopus Google Scholar). from the scanning that was a in between the ASOs (Fig. 5 induced IGF1R down-regulation in human ovarian and prostate cells, but was more in cell lines (Fig. and Thus, a major on siRNA in heteroduplex yield on the scanning we the of ASOs and siRNA on The relatively modest IGF1R down-regulation induced by ASOs or siRNA was not to to Akt (Fig. A and not transfection with induced profound IGF1R gene that we could not of the IGF1R or of a in the major downstream of the IGF1R (4Baserga R. Hongo A. Rubini M. Prisco M. Valentinis B. Biochim. Biophys. Acta. 1997; 1332: 105-126Crossref PubMed Scopus (480) Google Scholar). we the on and of transfection with 100 or siRNA and to strongly and weakly hybridizing The caused of and after 2 but this effect was with in cultures with the sequence oligonucleotide with siRNAs or inhibited of MDA-MB-231 for of each siRNA with inverted control with a effect in cells for with in cultures with the more siRNA duplex was of after 2 for comparisons with and These that the complex secondary structure of target transcripts can siRNA and that these to major in of potential We previously showed between and on gene by in of transcripts ASO (12Sohail M. Hochegger H. Klotzbucher A. Guellec R.L. Hunt T. Southern E.M. Nucleic Acids Res. 2001; 29: 2041-2051Crossref PubMed Scopus (57) Google Scholar). we have shown that of at 37 °C for target down-regulation within intact tumor This is the that were ASOs were at the 3′ and the of the not the Furthermore, hybridizations were performed in 1 was heteroduplex formation at a for IGF1R ASOs to in D.J. Biotechnol. 2000; PubMed Scopus Google Scholar) used a program designed to regions of the transcript duplex and structure D.J. Biotechnol. 2000; PubMed Scopus Google Scholar). of the selected ASOs within the region we targeting nt of the IGF1R transcript. This was to be and was shown to weakly on scanning with for 1 with the most ASOs IGF1R expression to of control D.J. Biotechnol. 2000; PubMed Scopus Google Scholar). We major in the activity of IGF1R by in the of of the ASOs toIGF1R mRNA. have two of of against human T. G. H. A. J. Biotechnol. 2002; 20: PubMed Scopus Google Scholar) and T. M. E. H. Nucleic Acids Res. 2002; 30: PubMed Scopus Google Scholar), in the to binding the siRNA target This is an for findings we in ASO and siRNA within intact cells and in RNase H The we between and siRNA suggest that secondary structure in the IGF1R transcript was a major factor in the of ASOs that induce RNase H siRNAs as to the complex to the homologous sequence in the target transcript S.M. 2001; PubMed Scopus Google Scholar). The nature of the molecular between siRNAs and target mRNA is The for with that for ASO binding the as was first S.M. 2001; PubMed Scopus Google Scholar, A. S. Nature. PubMed Scopus Google Scholar), of by between the transcript and of the This is with the that antisense strands can mediate in mammalian J. A. H. R. Tuschl T. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The for siRNA design is than the for ASOs C.A. J. Clin. Invest. 2001; 108: 641-644Crossref PubMed Scopus (157) Google Scholar, J. S.M. K. Tuschl T. Weber K. J. Cell 2001; PubMed Google Scholar). However a for could some siRNAs (13Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8018) Google Scholar) were to in cells, but in a expression was by siRNAs against regions of the transcript J. S.M. K. Tuschl T. Weber K. J. Cell 2001; PubMed Google Scholar). We have that mRNA folding has major for gene silencing, with implications for the design of Furthermore, the of these siRNAs in blocking IGF and tumor cell that IGF1R-targeting agents incorporating this mode of action have potential as anticancer therapy. We to Cancer for human IGF1R cDNA and to Research Laboratories, of for human insulin receptor