Poster abstracts
Poster number 53 submitted by Brianne Sanford
Modulating insulin receptor splicing as a potential therapeutic approach for rhabdomyosarcoma
Brianne Sanford (The Ohio State University Comprehensive Cancer Center, Columbus, OH 4321; Center for Childhood Cancer and Blood Diseases, The Research Institute at Nationwide Childrens Hospital, Columbus, OH, 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 (1Center for Childhood Cancer and Blood Diseases, The Research Institute at Nationwide Childrens Hospital, Columbus, OH, 43205; Department of Pediatrics, The Ohio State University, Columbus, OH, 43210)
Abstract:
The insulin receptor (IR) is subject to alternative splicing to produce two isoforms: full-length IR-B and an isoform lacking exon 11 known as IR-A, the predominant isoform expressed in sarcomas. This exon encodes for 12 amino acids and results in a receptor with high affinity for a growth hormone called insulin-like growth factor 2 (IGF2), which responds to autocrine and paracrine signaling. Increased IR-A levels observed in rhabdomyosarcoma (RMS) coupled with increased expression of IGF2, programs the tumor cell for enhanced growth and angiogenesis. Furthermore, IGF2 can maintain angiogenesis through IR-A when the cognate receptor for IGF2, the IGF-1 receptor, is blocked. Interestingly we have shown hypoxia increases alternative splicing to produce more IR-A. Adaptation to the hypoxic environment is a hallmark of the neoplastic phenotype. Therefore, we hypothesize that expression of splicing factors is altered under hypoxic conditions which leads to increased alternative splicing and generation of IR-A, allowing the cancer cells a growth advantage which contributes to metastasis beyond the micrometastatic phase.
To characterize sequence elements and splicing factors involved in the regulation of INSR alternative splicing, we have developed a hypoxia-inducible splicing system, which recapitulates the splicing patterns observed in tumors. We have shown that sequence elements preceding exon 11 are critical to the increased alternative splicing we see under hypoxic conditions. As such, we have targeted these regions for antisense oligonucleotide (ASO) development to increase IR-B, with the goal of restricting proliferative signaling by IGF2 activation of IR-A. We performed an ASO walk to target regions important for exon inclusion or exclusion and have successfully targeted a binding site for the splicing factor CUGBP1. RMS-derived cell lines almost exclusively express IR-A but when treated with our lead ASO compound we see a dramatic decrease in alternative splicing resulting in increased levels of IR-B. We have also shown that RMS-derived cells treated with our splice modulating ASO exhibit reduced cell proliferation and migratory properties. We postulate that modulation of INSR splicing can be used in conjunction with already established anti-IGF-1 receptor therapies to treat RMS.
Keywords: Splicing