Poster abstracts
Poster number 96 submitted by Xia Zhou
Bidirectional Antisense Control of Insulin Receptor Splicing Reveals Oncogenic and Metabolic Phenotypes in HCC Models
Xia Zhou (Department of Molecular Medicine and Therapeutics, The Ohio State University), Safiya Khurshid (Sanford Research ,University of South Dakota), Matias Montes (Department of Chemical and Systems Biology, Stanford University), Ashiwarya Jacob (Department of Biochemistry, University of Cambridge), Frank Rigo (Ionis Pharmaceuticals), Dawn Chandler (Department of Molecular Medicine and Therapeutics, The Ohio State University)
Abstract:
Alternative splicing of insulin receptor (INSR) exon 11 generates INSR-A and INSR-B, which preferentially support mitogenic and metabolic signaling, respectively. INSR-A is elevated in hepatocellular carcinoma (HCC) and responds to insulin and IGF-2 [1,3]. Here we show that splice-switching antisense oligonucleotides (SSOs) targeting distinct intronic regulatory elements bidirectionally reprogram INSR isoform expression and HCC-associated phenotypes.
SSO23 blocked an MBNL-binding enhancer in intron 11, promoting exon skipping and INSR-A production, whereas SSO55 blocked a CUGBP1-binding silencer in intron 10, promoting exon inclusion and INSR-B [2]. In HCC cells, SSO55 reduced proliferation and migration, whereas SSO23 enhanced migration. Quantitative immunofluorescence showed that SSO23 increased IGF-2-induced AKT phosphorylation, while SSO55 reduced p-AKT, linking INSR isoform selection to differential pathway activation.
To model this splicing program in vivo, we generated CRISPR-engineered mice carrying mutations in the intron 11 MBNL-binding region targeted by SSO23. These mice exhibited increased hepatic Insr-A and liver pathology. Metabolic profiling of aged PAM mutants revealed heterogeneous, sex-dependent changes, including increased expression of lipogenic genes, particularly Scd1, altered hepatic triglycerides and cholesterol, and reduced Ldlr, Lrp1, Mttp and Hmgcr in sex-specific patterns. These data associate increased Insr-A with altered lipid synthesis, clearance and lipoprotein export, although the underlying mechanism remains to be established. PAM and DM mutants and wild-type littermates were also subjected to an HCC-promoting Western diet, with body weight, serum AFP, liver-function markers and histopathology monitored through 22 weeks to determine whether altered Insr splicing increases susceptibility to diet-induced HCC.
Our findings identify INSR exon 11 as an actionable RNA-processing node connecting oncogenic signaling with hepatic lipid homeostasis. Selectively restoring INSR-B may constrain cancer-associated INSR-A activity while preserving metabolic receptor function, providing a framework for isoform-directed RNA therapy in HCC.
References:
1. Belfiore A et al. Insulin receptor isoforms in physiology and disease. Endocr. Rev. 38, 379431 (2017).
2. Paul S et al. Interaction of muscleblind, CUG-BP1 and hnRNP H proteins in DM1-associated aberrant IR splicing. EMBO J. 25, 42714283 (2006).
3. Sen S et al. Deletion of serine/arginine-rich splicing factor 3 in hepatocytes predisposes to hepatocellular carcinoma in mice. Hepatology 61, 171183 (2015).
Keywords: INSR alternative splicing, splice-switching oligonucleotides, hepatocellular carcinoma