Study of cell cycle targets critical to the proliferative response initiated by the multiple endocrine neoplasia type II A oncogene - RET2A
Multiple endocrine neoplasia type II A (MEN2A), characterized by medullary thyroid carcinoma, adrenal medullary pheochromocytoma and parathyroid hyperplasia, is predisposed by activating mutations in the RET proto-oncogene. Activated RET, or its constitutively active MEN2 mutant (RETC634R - RET2A), stimulates several well-characterized signal transduction pathways. However, the basic underlying mechanism of RET-mediated tumorigenesis remains poorly understood. Interestingly, mice deficient for both Cyclin Dependent Kinase Inhibitors (CDKIs), p18 and p27, develop a tumor spectrum which overlaps that observed in MEN patients. The purpose of this study, therefore, was to investigate whether RET in its activated form, regulates the expression of p18 and p27. We demonstrate that induced i expression of RET2A, or ligand-dependent activation of wild type RET, correlates with a p42/44 mitogen-activated protein (MAP) kinase-dependent decrease in both protein and mRNA levels of p18 and p27 under conditions of cellular growth arrest, where these CDKIs would normally be elevated. RET2A expression also correlates with decreased p27 protein stability. Furthermore, RET activation leads to an inappropriate induction of D type cyclins, which occurs independently of the MAP kinase pathway. These changes in cell cycle proteins is accompanied by increased Retinoblastoma protein phosphrylation and subsequent cell proliferation, which is inhibited by blocking the decrease in p18 and p27 using a MEK (MAPK ERK kinase) inhibitor, PD98059. Furthermore, we show by using p18 siRNA, that the decrease in p18 is required and sufficient for RET2A-mediated proliferation. Perhaps, most significantly, MEN2A adrenal tumors also display these changes in cell cycle expression profile, demonstrating the biological relevance of our cell culture studies. Our results demonstrate for the first time that RET2A regulates p18, and suggest that loss of not only p27 but also of p18 expression is a key step in MEN tumorigenesis. Lastly, we demonstrate differential effects of RET activation on the cell cycle proteins depending on the nature of the ligand/coreceptor complex that RET is activated with. Based on these results, we propose a model to explain the tissue specificity during MEN2 tumorigenesis. In conclusion, these studies expand our understanding of RET function, CDKI regulation and cell cycle control during MEN2 tumorigenesis.
Franklin, Purdue University.
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