Retinoic Acid Induces REST Degradation and Neuronal Differentiation by Modulating the

BACKGROUND: The repressor element-1 silencing transcription factor (REST) is a repressor of neuronal genes. Its expression is associated with poor neuronal differentiation in many neuroblastoma patient samples and cell lines. Because retinoic acid promotes neuronal differentiation, the authors postulated that it involves modulation of REST expression. METHODS: The expression of REST and of an S-phase kinase-associated protein 1/cullin 1/F-box (SCF) protein complex that contains the F-box protein b-transducin repeat-containing protein (b-TRCP) (SCF b-TRCP ) in neuroblastoma tumor samples and cell lines was analyzed by immunofluorescence and Western blot analysis. SK-N-SH and SK-N-AS cells were treated with retinoic acid and MG-132 to measure proteasomal degradation of REST by Western blot and quantitative real-time polymerase chain reaction analyses. Immunoprecipitation and coimmunoprecipitation assays were done in SKN-AS cells that were transfected either with a control plasmid or with an enhanced green fluorescent protein-SCF b-TRCP – expressing plasmid. RESULTS: Several neuroblastoma patient samples and cell lines displayed elevated REST expression. Although, REST transcription increased upon retinoic acid treatment in SK-N-SH and SK-N-AS cells, REST protein levels declined, concomitant with the induction of neuronal differentiation, in SK-N-SH cells but not in SK-N-AS cells. MG-132 treatment countered the retinoic acid-mediated decline in REST protein. SCF b-TRCP , a known REST-specific E3-ligase, was poorly expressed in many neuroblastoma samples, and its expression increased upon retinoic acid treatment in SK-N-SH cells but declined in SK-N-AS cells. Ectopic expression of SCF b-TRCP in SK-N-AS cells promoted REST ubiquitination and degradation and neuronal differentiation. CONCLUSIONS: The current results indicated that elevated transcription of REST compounded by its impaired degradation by SCF b-TRCP may contribute to the failure of these tumors to differentiate in response to retinoic acid. Cancer 2011;117:5189–202. V C 2011 American Cancer Society.