Abstract 5826: Essential role for cyclic-AMP responsive element binding protein 1 (CREB1) phosphorylation in the survival of medulloblastoma patients
Authors: Zomerman Walderik W. , Plasschaert Sabine L. , Scherpen Frank J. , Lourens Harm Jan J. , Huizinga Geesina C. , Hoving Eelco W. , Dunnen Wilfred F. den , Bruggeman Sophia , Bont Eveline S.J.M. de 
Published: July 2017

Prognosis for recurrent medulloblastoma patients is extremely poor, with a median survival of 26.8 months. Treatment options for recurrent medulloblastoma patients are limited and ineffective due to resistance to chemotherapeutic agents and radiotherapy. Therefore, more research is required to address the high mortality of medulloblastoma patients that have therapy-resistant medulloblastoma. In a previous, study, we showed that cyclic-AMP responsive element binding protein (CREB1) was highly phosphorylated in primary medulloblastoma specimens. CREB is an important transcription factor involved in cerebellar cell proliferation and survival. In various cancers types, the expression of CREB is associated with prognosis. Untill now, CREB expression and its relation with overall survival has not been studied in medulloblastoma patients. In this study, we determined CREB phosphorylation levels and mRNA levels of CREB and its transcriptional co-activators CREBBP and EP300 using peptide phosphorylation and gene expression arrays in a cohort of 50 pediatric medulloblastoma patients. Furthermore, we performed shRNA-mediated knockdown of CREB, CREBBP and EP300 mRNA levels in medulloblastoma cell line DAOY. Knockdown was confirmed by means of qRT-PCR and western blotting. Next, cell viability assays were used to investigate the effect of chemotherapeutic reagents (etoposide and/or cisplatin) on cell viability in CREB, CREBBP or EP300 knockdown cells or in combination with a small molecule CREB inhibitor (KG-501).Low CREB phosphorylation levels in combination with low CREBBP and EP300 mRNA levels was significantly associated with poor overall survival (p=0.0001) with a median survival of 11.7 months in medulloblastoma patients. High CREB phosphorylation in combination with high CREBBP and EP300 mRNA levels was associated with a favourable prognosis (5 year-overall survival: 91%). Supervised clustering of gene expression data based upon low versus high CREB phosphorylation and CREBBP/EP300 mRNA levels and subsequent gene ontology analysis showed significant enrichment of genes associated with cerebellar and neuronal differentiation and development. Furthermore, knockdown with shCREB.3, shCBP.1-2 or shEP300A completely rescued DAOY cells from the etoposide-induced decrease in cell viability that was observed in the scrambled control. Similarly, addition of the CREB inhibitor KG-501 (2μM) completely rescued DAOY cells from the etoposide-induced decrease in cell viability and partially rescued DAOY cells from the cisplatin-induced decrease in cell viability.Together, these findings support an important role for CREB in neuronal differentiation, chemosensitivity and in the overall survival of pediatric medulloblastoma patients.

Citation Format: Walderik W. Zomerman, Sabine L. Plasschaert, Frank J. Scherpen, Harm Jan J. Lourens, Geesina C. Huizinga, Eelco W. Hoving, Wilfred F. den Dunnen, Sophia Bruggeman, Eveline S.J.M. de Bont. Essential role for cyclic-AMP responsive element binding protein 1 (CREB1) phosphorylation in the survival of medulloblastoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5826. doi:10.1158/1538-7445.AM2017-5826