Abstract B39: Modulation of insulin receptor alternative splicing to develop cancer therapeutics

Abstract Insulin receptor (IN-R) undergoes alternative splicing to produce two isoforms: the full-length IN-RB and exon 11 skipped IN-RA isoform. While IN-RB has high affinity for insulin and much lower affinity for IGF1 or IGF2 ligands, IN-RA binds both insulin and IGF2 with high affinity and as such it exploits the IGF pathway to accelerate the onset of tumor-cell hallmarks such as proliferation and angiogenesis. Our data show that there is a significantly increased expression of IN-RA levels in multiple cancer patient cohorts, including rhabdomyosarcoma (RMS) and osteosarcoma (OS). We also found this increased IN-RA expression in tumor cell lines derived from multiple different pediatric tumor types such as RMS, OS, and Ewing’s sarcoma as compared to the control samples. Additionally, our data show that cellular stress such as hypoxia increases the expression of the IN-RA isoform. Adaptation to hypoxic environments is a hallmark of the neoplastic phenotype, and pediatric sarcomas exhibit an intrinsically hypoxic physiology. However, how the INR alternatively spliced mRNAs are regulated is not well understood, nor is the role that these isoforms play in the initiation and progression and therapeutic resistance of human cancer. Our central hypothesis is that hypoxia alters the expression of splicing factors, leading to the generation of the IN-RA isoform, which contributes to cancer progression beyond the micrometastatic stage. We used an insulin receptor mini-gene system to identify a binding site for the RNA binding protein cugbp, critical for the hypoxia-induced splicing regulation of IN-R. We targeted the cugbp binding site using an antisense oligonucleotide (ASO) walk to identify an ASO that binds to the cugbp binding site in the intron 10 of INR gene and modulates the levels of endogenous IN-RA. To allow the possibility for therapeutic intervention, we utilized this ASO in OS cell lines. We found that the application of ASO shifts the IN-R splicing towards IN-RB and significantly reduces proliferation, migration, and angiogenesis in cancer cell lines. In order to delineate the downstream consequences of this splicing change, we subjected the control and ASO-treated cells to mass spectrometric analysis. Preliminary analysis of this data reveals that proliferation markers such as Ki67, PI3-kinase pathway components, protein clusters such as cell-cell adhesion, and cell division, among others, are significantly downregulated in cells treated with ASO where the splicing has been restored to the IN-RB isoform. Our data open a new paradigm of how alternative splicing regulates cell signaling to mediate cancer-causing changes, and our ASO compounds show promising insight into how we can reinstate the splicing pattern and impede tumorigenesis. Citation Format: Safiya Khurshid, Matias Montes, Brianne Sanford, Peter Houghton, Frank Rigo, Dawn Chandler. Modulation of insulin receptor alternative splicing to develop cancer therapeutics [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B39.

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