The role of MDM2-p53 axis in pathogenesis of embryonal brain tumors

Abstract

During nervous system development, neural stem cells (NSCs) and their derivative progenitor cells undergo a proliferative expansion prior to differentiation and migration to their final destination. Aberrations in this tightly controlled process that shift the balance from differentiation toward proliferation is implicated in the pathogenesis of embryonal brain tumors including medulloblastoma (MB), the most common malignant brain tumor in children. Despite advances in current therapeutic strategies, young patients with embryonal brain tumors retain life-long post-treatment complications due to lack of treatment specificity and toxicity to developing nervous system. A better tumor-targeted therapy for embryonal brain tumors is critically needed. Activation of the p53 tumor suppressor in embryonal brain tumor might have therapeutic potentials as these tumors rarely carry p53 mutation. Our laboratory has previously demonstrated that MDM2, a key negative regulator of p53, is required for MB tumorigensis. Accordingly, reduction of the MDM2 level induces apoptosis in a subset of cerebellar granular neuronal progenitors (GNPs) in a p53-dependent manner. Our previous findings further suggested that GNPs lacking MDM2 are also prone to premature differentiation. In current study, we elucidated the inhibitory role of MDM2 on p53 in regulation of apoptosis and differentiation of premature neuronal cells. Given that MB tumors contain undifferentiated neuronal cells, first, the biological outcome of MDM2 inhibition by small molecule, nutlin-3 was studied in a panel of human MB cells. Nutlin-3 appears to release p53 inhibition from MDM2 in human MB cells harboring wild-type TP53 and thereby, induces p53 target genes leading to apoptosis and/or cell cycle arrest. Nutlin-3 potentiates anti-proliferative effects of doxorubicin inTP53 wild-type MB cells through enhancement of p53 function. In contrast, TP53 mutant MB cell lines are highly resistant to nutlin-3 treatment. However, at high concentrations, nutlin-3 could induce apoptosis in TP53 mutant MB cell suggesting that nutlin-3 may also be effective in regression of TP53 mutant MB tumors through an alternative mechanism. Next, to gain a better insight in mechanism(s) by which MDM2 determines cellular fate toward differentiation or apoptosis, regulatory role of MDM2 in neuronal differentiation was elucidated in anin vitro model employing the human embryonal teratocarcinoma NT2/D1 cell line. NT2/D1 cells can differentiate to mature neurons in response to retinoic acid (RA). Induction of neuronal differentiation in these cells resulted in activation of p53 concomitant with a decrease in the level of MDM2. Accordingly, inhibition of MDM2 by nutlin-3 promoted differentiation of NT2/D1 to mature neurons as shown by up-regulation of neuronal markers, revealing that release of p53 inhibition from MDM2 promotes neuronal differentiation. The reduction of MDM2 in differentiating NT2/D1 cells was at the protein level and mediated by proteasomal degradation. In keeping with the importance of ubiquitination as an obligatory step for proteasomal degradation of proteins, a change in the levels of several enzymes that regulate MDM2 stability via ubiquitin-mediated mechanisms was observed in differentiating NT2/D1 cells. In these cells, MDM2 deubiquitinating enzymes, USP2a and HAUSP were down-regulated while the ubiquitinating enzyme, PCAF was up-regulated. Together, my findings suggest that proteasomal degradation of MDM2 and the subsequent activation of p53 may play a role in promoting the neuronal differentiation. This further provides the new insights regarding the potential molecular mechanism that may provoke prodifferentiation function of MDM2-p53 axis. Overall, my findings identify MDM2 as a potential target to suppress the growth of TP53 wild-type embryonic brain tumors through the induction of either differentiation or cell death