Alternative pre-messenger RNA (pre-mRNA) splicing has emerged as a predominant mechanism for proteomic diversity and gene expression. This diversity has been emphasized by recent work that elucidates a network of alternatively spliced genes in response to cell stimuli that are part of the tumorigenic process (DNA damage and Epithelial to Mesenchymal Transition, EMT, for example). Though the importance of alternative splicing in a number of cell processes has been recognized since its discovery, the mechanisms underlying its regulation remain poorly understood. Solid tumors are characterized by both transient and chronic hypoxia, and the ability for tumor cells to adapt to hypoxia is essential for tumor progression. We have shown recently that stimulation of vascular endothelial cell proliferation and angiogenesis induced by the major angiogenic factor, VEGF, is dependent upon insulin-like growth factor (IGF) signaling. Additionally, both human vascular endothelial cells and cells derived from pediatric sarcomas express predominantly the alternatively spliced form of the Insulin Receptor gene (IN-R), that has high affinity for IGF-2. Importantly, IN-R is spliced in response to hypoxia, and pediatric sarcoma cells secrete IGF-2. Consequently, IN-R splicing appears critical in progression of pediatric sarcomas both from acting as a receptor for autocrine growth of tumor cells, and for paracrine growth of vascular cells, and hence angiogenesis. The primary goal of this proposal is to understand the mechanisms and consequences of IN-R pre-mRNA splicing in response to hypoxia. This work will test the hypothesis that regulatory elements and splicing factors are modulated in response to hypoxia and are involved in the alternative splicing of IN-R to contribute to tumorigenesis. In order to determine the effectors of IN-R hypoxia- induced splicing, we will be screening hypoxia induced splicing using a cellular assay which robustly recapitulates changes in IN-R splicing that are seen in cancer cells. We will utilize the hypoxia-induced splicing assay to identify RNA sequences and their respective binding partners that are necessary for regulation of IN-R pre-mRNA splicing. Furthermore, we will use established mouse xenograft assays and novel antisense oligonucleotides (ASOs) to modulate IN-R splicing and determine the role of the spliced isoforms in tumor progression and as novel targets for therapeutic intervention.
Recent advances in research have uncovered a surprising link between alternative splicing and tumor development, although the mechanisms of alternative splicing are not understood. By understanding these mechanisms, we can gain insight into how induced alternative splicing may affect tumor development and, ultimately, identify new intervention points for cancer treatment. In this proposal, we investigate targeting splicing of the insulin receptor for treatment of pediatic rhabdomyosarcoma.
