P-05 Specific targeting of IGF-I and IGF-IR genes by antigene nucleic acids

Type I insulin-like growth factor (IGF-I) and its receptor (IGF-IR) have been implicated in progression of a large number of cancers, such as glioblastoma, hepatocarcinoma, prostatic and breast carci- nomas. Because of their central role in oncogenic maintenance and metastasis processes, the IGF axis molecules represent important targets for anti-cancer strategies. Recently, tyrosine kinase inhibitors and antibodies have been used to block the action of IGF-I and its receptor. Numerous nucleic acid-based strategies, including antisense RNA, antisense oligonucleotides, ribozymes, triplex-forming oligori- bonucleotides, short hairpin RNA and short interfering RNA, have also been developed to block IGF-I and IGF-IR expression. At present, we will describe an innovative approach to inhibit the func- tions of IGF-I and its receptor using triplex-forming oligonucleotides (TFO) targeted to oligopyrimidine-oligopurine double-stranded se- quence present in both genes: in IGF-I promoter region and in intron 2 of IGF-IR. We first demonstrated that IGF-I TFO very slightly inhibited reporter gene expression under the control of the rat IGF-I promoter in an hepatocarcinoma stable cell line, while IGF-IR TFO did not inhibit IGF-IR transcription as measured by real-time RT-PCR. Both TFOs were then conjugated to a well-known topoisomerase I inhibitor, camptothecin (CPT), and were shown to induce in vitro DNA cleavage specifically at the triplex binding site, due to the poisoning of topoisomerase I at their binding site. We have shown that these CPT- TFO conjugates selectively and efficiently inhibited the expression of the targeted endogenous genes in cells. Moreover, this inhibitory effect was clearly mediated by topoisomerase I as demonstrated upon silencing of the enzyme in cancer cells by RNA interference and upon use of a camptothecin-resistant cell line. Localized cleavage of DNA mediated by these TFOs was suggested by the analysis of phospho- H2AX foci in CPT-TFO transfected tumour cells. We will also present preliminary results obtained with other nucleic-acid based approaches.