Abstract 2900: Sumoylation of the retinoic acid receptor alpha protein represses transcriptional activity and contributes to retinoic acid resistance in glioma stem cells

Abstract

Abstract Introduction: Glioblastoma (GBM) is the most deadly form of brain cancer. Despite treatment with tumor resection followed by radiation and chemotherapy, the median survival rate is less than 15 months. Glioma stem-like cells (GSCs) are thought to be treatment resistant and drive tumor growth. Retinoids have been successfully used to terminally differentiate the cancer stem cell population in some leukemias, but show only modest success in GBM despite the presence of RARA in tumor cells. Resistance to retinoic acid (RA) can develop due to mutations in the retinoic acid receptor alpha (RARA) protein that prevent its phosphorylation. This study evaluated the retinoic acid receptors to determine if receptor dysfunction is the underlying cause of resistance in GBM. Methods: Several GSCs derived from both primary (GSC923) and recurrent (GSC827, GSC604) GBM tumors and normal mouse neural stem cells (MNSC) were analyzed for RARA protein expression by Western blots using an RARA specific antibody. 2D Westerns were used to measure RARA phosphorylation in response to RA treatment. MNSC were treated with RA in the presence or absence of MG132, a proteasomal inhibitor, to determine if MNSC RARA protein was sensitive to proteasomal degradation. RARA transcriptional activity was measured using a promoter luciferase construct containing a specific retinoic acid response element (RARE). Immunoprecipitations using either Sumo1, Sumo 2/3 or RARA identified the type of sumo peptide covalently attached to the endogenous GSC RARA protein. Results: MNSCs express the predicted 51 kDa RARA protein; however, GSC827 and GSC923 both express two high molecular weight forms that were approximately 62 and 73 kDa. In response to RA, the MNSC RARA became phosphorylated, but the GSC827 RARA remained hypophosphorylated. Treatment with RA induced the proteasomal degradation of MNSC RARA as expected. In contrast, both forms of the GSC RARA proteins were resistant to ligand-induced proteasomal degradation. In response to RA, the RARA protein in three glioma stem cell lines showed a 40-60% decrease (p = 0.008) in transcriptional activity compared to MNSC. Immunoprecipitation showed that the Sumo1 peptide is covalently attached to the GSC RARA protein, occurring before the treatment with retinoic acid. Conclusions: We demonstrated that aberrant posttranslational modification of the RARA protein contributes to retinoic acid resistance in glioma stem cells. Covalent attachment of the Sumo1 peptide likely prevents RARA phosphorylation and ligand-induced proteasomal degradation. The Sumo1 peptide serves as a scaffold to bind nuclear corepressor proteins. Future exploration of the role of the Sumo1 modification may uncover underlying mechanisms of inherent retinoic acid resistance in GSCs potentially leading to successful differentiation therapy in GBM. Citation Format: Virginia W. Rodriguez, Rolanda Bailey, Mark Gilbert. Sumoylation of the retinoic acid receptor alpha protein represses transcriptional activity and contributes to retinoic acid resistance in glioma stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2900.