N-Myc—A critical target of the proliferative response initiated by the multiple endocrine neoplasia type II A oncogene - Ret2A

Human Multiple endocrine neoplasia type II A (MEN2A) is predisposed by inherited activating mutations in the RET proto-oncogene. Ligand and coreceptor-mediated dimerization of RET leads to activation of numerous signal transduction pathways however, the tumorigenic mechanism of the MEN2A specific, constitutively active, mutant RETC634R (RET2A) is poorly understood. Interestingly, RET2A expression correlates with an increase in cyclin D1/D2 and a decrease in Cyclin Dependent Kinase Inhibitors (CDKIs), p18 and p27. Moreover, repression of p18 is a rate-limiting step for RET2A-mediated hyperproliferation. Therefore, this study was designed to investigate the molecular mechanisms through which RET2A regulates p18 expression. We demonstrate that induced in vitro expression of RET2A correlates with an increase in cyclin D1 and a concomitant p42/44 mitogen-activated protein (MAP) kinase-dependent decrease in mRNA levels of p18 and p27 under conditions of cellular growth arrest, where these CDKIs would normally be elevated. RET2A expression also leads to a MAPK-dependent induction of N-Myc mRNA and protein levels. The induction in N-Myc is concurrent with the increase in Cyclin D1/D2, but precedes the decrease in p18 and p27 levels. We also demonstrate that the MAPK-dependent increase in N-Myc is required for RET2A-mediated repression of both p18 and p27, but not for induction of Cyclin D1/D2. N-Myc expression and the sequences within the p18 promoter region containing the N-Myc binding site are required for RET2A-mediated p18 repression. Induction in N-Myc expression results in elevated binding to its consensus binding site within the p18 promoter. Finally, cells that express RET2A but lack N-Myc lose the proliferative advantage offered by RET2A, supporting the critical role of N-Myc as the mediator of RET2A signals to p18, p27 and the cell cycle. Our results demonstrate for the first time that RET2A regulates N-Myc, and suggest that induction of N-Myc is a key step in MEN tumorigenesis. Based on our results, we propose a model to explain the roles of RET, N-Myc and the CDKIs p18 and p27 during normal development and the endocrine tissue specificity of MEN2A tumors