MBP-1 represses N-MYC expression and acts as a tumor suppressor in human Neuroblastoma LAN-5 cells

Abstract

Neuroblastoma is the most common extra-cranial solid tumor of childhood, originated from cells of the neural crest. Amplification of N-MYC gene and 1p-deletion are found in more than 30% of patients with advanced stages and they are associated with poor prognosis. An alternative translated product of the ENO1 gene, known as MBP-1 (c-myc promoter binding protein-1), acts as a negative regulator of the c-MYC oncogene, ERBB2 and COX-2 genes, furthermore, ENO1/MBP-1 overexpression in Neuroblastoma cells significantly reduces cell growth and induces apoptosis. Even though there are similarities between the c-MYC and N-MYC oncogenes, there are no evidences that MBP-1 is able to interact with N-MYC promoter to regulate negatively its expression. Reporter plasmids (luciferase), containing various portions of N-MYC promoter, were used in N-MYC-amplified Neuroblastoma cells (LAN-5) transfection experiments with an effector plasmid expressing Flag-MBP-1 protein. These studies have identified a promoter region involved in the negative regulation of N-MYC by MBP-1, and its binding activity, within the N-MYC promoter was confirmed, both in vitro and in vivo, by EMSA and chromatin-immunoprecipitation (ChIP) assays. SiRNA-mediated induction of endogenous MBP-1 expression in LAN-5 cells determines a decrease of N-MYC and a significant increase of p21, BAX and γ-enolasi expression (a marker of neuronal differentiation). Furthermore, migration and cytotoxicity assays indicate that MBP-1 induces apoptosis and reduces the migration of LAN-5 cells. In conclusion, the data presented indicate that MBP-1 acts as a transcriptional repressor of N-MYC gene and its expression induces senescence, apoptosis and differentiation in LAN-5 Neuroblastoma cells.