Poster abstracts

Poster number 34 submitted by Daniel Comiskey

MDM2 Alternative Splicing in Cancer: Identifying Molecular Mechanisms

Daniel F. Comiskey, Jr. (Department of Pediatrics, The Ohio State University), Ravi K. Singh (Department of Pediatrics, The Ohio State University), Aixa S. Tapia-Santos (Department of Pediatrics, The Ohio State University), Dawn S. Chandler (Department of Pediatrics, The Ohio State University)

Abstract:
The proto-oncogene MDM2 is an E3 ubiquitin ligase and chief negative regulator of the tumor suppressor protein p53. In cancer and in response to stress, MDM2 undergoes alternative splicing. One such splice variant, MDM2-ALT1, contains only exons 3 and 12, leads to the stabilization of p53, and subsequently cycle arrest and/or apoptosis. Paradoxically, expression of mouse homolog Mdm2-b in mice promotes tumorigenesis. The regulation of MDM2 alternative splicing is not well characterized and it is therefore critical to understand the regulation of stress-induced MDM2 alternative splicing to identify novel targets for anticancer therapies.

To study the alternative splicing of MDM2 we have developed an in vitro splicing system using MDM2 minigenes and normal and cisplatin-treated HeLa S3 nuclear extracts. The MDM2 3-11-12 minigene predominantly excludes exon 11 under UV and cisplatin treatment in vivo and in vitro, recapitulating the behavior of the endogenous gene. We identified predicted binding sites for splicing regulators SC35 and SF2/ASF in exon 11 of the MDM2 minigene and made mutations in their binding sites to uncover their roles in splicing of MDM2.

We confirmed the affinity of these proteins for their targets by RNA pull downs. Mutation of predicted sites led to exon inclusion in both in vitro splicing assays and in cell transfections. Overexpression of SF2/ASF in MCF-7 cells led to skipping of exon 11 in our wild-type MDM2 minigene, while the corresponding mutant was impervious to SF2/ASF expression. When we knocked down expression of SC35 in SC35 Tet-Off mouse embryonic fibroblasts our wild-type MDM2 minigene excluded exon 11 and the corresponding mutants did not. Overall, our results provide insight into the regulation of damage-induced MDM2 alternative splicing by SC35 and SF2/ASF, presenting potential targets for therapeutic strategies.

Keywords: MDM2, Alternative Splicing, Cancer