Abstract 2025: REST and SCFβ-TRCP expression determine the response of neuroblastoma cells to retinoic acid

Abstract

Abstract BACKGROUND: Retinoic acid (RA) is a mainstay of neuroblastoma treatment as it promotes tumor differentiation. However, the mechanism underlying RA-mediated neuronal differentiation is not well understood. The purpose of this study is to better define the molecular targets through which RA promotes differentiation in neuroblastoma cells. The RE-1 Silencing Transcription Factor (REST) is a repressor of neuronal genes. It is expressed in embryonic stem cells and neural progenitors, and is downregulated during neurogenesis. REST is overexpressed in many neuroblastoma tumor samples and cell lines, and its expression is associated with an undifferentiated phenotype. Since REST is critical for neuronal differentiation, we hypothesized that RA promotes differentiation in neuroblastoma cell lines by downregulating REST expression. METHODS AND RESULTS: Quantitative reverse transcription polymerase chain reaction, immunofluorescence assay and western blotting revealed REST expression to be elevated in a number of human neuroblastoma tumors and cell lines. Treatment with RA promoted neuronal differentiation in some but not all cell lines. In RA-responsive cells, induction of neuronal differentiation was associated with a decline in REST protein levels and upregulation of REST-target gene expression. However, REST protein levels were elevated upon RA treatment in resistant cells, REST transcript was upregulated in both RA-sensitive and resistant cells as expected. These results suggested that RA downregulated REST through post-transcriptional mechanisms, which was confirmed by measurement of REST protein levels in the presence of cycloheximide. Treatment with MG-132 blocked RA-mediated degradation of REST, indicating the involvement of the proteasome. The E3-ligase, SCFβ-TRCP, is known to be involved in REST ubiquitination and degradation. Low levels SCFβ-TRCP were associated with elevated REST protein in a number of human neuroblastoma tumors and cell lines. We observed that SCFβ-TRCP transcript and protein were elevated upon RA-treatment in responsive cell lines but not in RA-resistant cells with high REST levels. Ectopic expression of SCFβ-TRCP promoted REST ubiquitination and degradation in RA-resistant cells and formed a complex with ubiquitinated REST as determined by immunoprecipitation and co-immunoprecipitation experiments. Since these events were associated with induction of neuronal differentiation, our findings suggest that a failure to degrade REST blocks neuronal differentiation upon RA treatment. CONCLUSION: Our study indicates that deranged proteasomal machinery may contribute to maintenance of REST expression and the blockade of neuronal differentiation in neuroblastoma cells. Agents with potential to upregulate SCFβ-TRCP expression may have therapeutic value in patients that fail to respond to RA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2025. doi:10.1158/1538-7445.AM2011-2025