Poster abstracts
Poster number 2 submitted by Brianne Sanford
Modulation of insulin receptor splicing to develop cancer therapeutics
Brianne Sanford (Center for Childhood Cancer and Blood Diseases, The Research Institute at Nationwide Childrens Hospital, Columbus, Ohio, 43205), Chelsea Brown, Hemant Bid, Thomas Bebee, Daniel Comiskey Jr. (Center for Childhood Cancer and Blood Diseases, The Research Institute at Nationwide Childrens Hospital, Columbus, Ohio, 43205), Frank Rigo (Ionis Pharmaceuticals, Carlsbad, California, 92010), Peter Houghton (Greehey Childrens Cancer Research Institute, University of Texas Health Science Center, San Antonio, Texas, 78229), Dawn Chandler (Center for Childhood Cancer and Blood Diseases, The Research Institute at Nationwide Childrens Hospital and Department of Pediatrics, OSU Columbus, Ohio, 43210)
Abstract:
The insulin receptor (IN-R) alternatively spliced variant A, encoding a receptor lacking exon 11, is the predominant isoform expressed in both pediatric sarcoma cells and vascular endothelial cells. Exon 11 of the IN-R gene is composed of 36 nucleotides that encode 12-amino acids that reside at the carboxyl terminus of the alpha subunit. Though only a small change in the protein composition, deletion of these 12 amino acids results in a receptor that has increased binding affinity for the IGF-2 growth factor and is exquisitely capable of responding to autocrine and paracrine signaling. Cells expressing both the IN-R A variant and the IGF-2 growth factor are therefore programmed for growth and angiogenesis, two essential steps in tumorigenesis. Furthermore, the IGF-2 can maintain angiogenesis through IN-R A when the IGF-1 receptor is blocked. We also demonstrate that hypoxia induces IN-R alternative splicing. This finding is particularly intriguing since recent studies have shown that adaptation to hypoxic environments is a hallmark of the neoplastic phenotype. We therefore hypothesize that the expression of splicing factors is altered by hypoxia leading to the generation of the IN-R A isoform, which contributes to cancer progression beyond the micrometastatic stage.
To characterize sequence elements and RNA binding proteins in the regulation of IN-R alternative splicing, we have designed a hypoxia-induced splicing system that recapitulates the IN-R splicing seen in tumors. We have shown that sequence elements flanking the regulated exon are important for the hypoxia-induced splicing changes. Furthermore, we have performed an ASO walk to target these splicing enhancer and repressor sequences to interfere with IN-R splicing. We have successfully targeted the CUGBP1 negative regulator of splicing that resides in intron 10 to increase the IN-R B full-length isoform. We have shown that an ASO used to drive production of the IN-R B isoform in a rhabdomyosarcoma (RMS) cell line, which normally expresses the IN-R A isoform, affects cancer cell behavior to induce reduced motility and cell migration. We therefore postulate that modulation of the splicing of the IN-R, alone or in conjunction with IGF-1R blocking antibodies, could be used as a therapeutic intervention in pediatric RMS cells.
Keywords: splicing, cancer, antisense oligonucleotide