Splice-switching of the insulin receptor in rhabdomyosarcoma: Rescuing theIR-Bisoform for better treatment options

Abstract

ABSTRACTRhabdomyosarcoma (RMS) is an aggressive pediatric tumor with poor prognosis for metastasis and recurrent disease. Large scale sequencing endeavors demonstrate that RMS tumors have limited mutations and a dearth of driver mutations that are precisely targetable. However, IGF2 signaling is known to be grossly altered in RMS. The IGF2 signalling molecule binds both its innate IGF1 receptor as well as the insulin-receptor-variant-A (IR-A) with high affinity. Mitogenic and proliferative signalling via the canonical IGF2 pathway is therefore augmented byIR-A. The insulin receptor (IR) which is a transmembrane tyrosine-kinase receptor exists in two alternatively spliced isoforms,IR-AandIR-B. In this study, we show that RMS patients express increasedIR-Acompared to control tissues that express predominantly theIR-Bisoform. We also found thatHif1ais significantly increased in RMS tumors, portraying their hypoxic phenotype. Furthermore, the alternative-splicing ofIRadapts to produce moreIR-Ain response to hypoxic stress. Upon examining the pre-mRNA structure of the gene, we identified a hypoxia-responsive-element, which is also the binding site for the RNA-binding proteinCUG-BP1. We designed Splice-Switching-Oligonucleotides (SSO) against this binding site to decrease the levels ofIR-Ain RMS cell-lines and consequently rescue theIR-Bexpression levels. SSO treatment resulted in significant reductions in proliferation, migration and angiogenesis. Our data show promising insight into how impeding the IGF-2 pathway by reducingIR-Aexpression mitigates tumor growth. Our data reveal that RMS tumors useIRalternative-splicing as yet another survival strategy which can be exploited as therapeutic intervention in conjunction with already established anti-IGF-1 receptor therapies.