MYCN and the p53-MDM2/MDMX-p14ARF network in neuroblastoma amd response to MDM2-p53 antagonists

Abstract

Background: MYCN-amplification is a major negative prognostic marker, occurring in 25-30% of neuroblastomas. MYCN plays contradictory roles in promoting cell growth and sensitizing cells to apoptosis, and we have recently shown that p53 is a direct transcriptional target of MYCN, and may be an important mechanism of MYCN-induced apoptosis. Although p53 mutations are rare in neuroblastoma at diagnosis, the p53/MDM2/p14ARF pathway is inactivated in 35% of cases through MDM2-amplification or p14ARF inactivation. Neuroblastoma is therefore an ideal target for p53 reactivation using MDM2-p53 antagonists. MDMX, a homologue of MDM2, is another negative regulator of p53 which is often overexpressed in cancers and has been shown to compromise the effects of MDM2-p53 antagonists in various cancer types. MDMX expression and the effect on MDM2-p53 antagonists has not been investigated in neuroblastoma. Hypotheses 1) Reactivation of p53 by inhibition of its negative regulator MDM2, using the MDM2-p53 antagonists Nutlin-3 and MI-63, will result in p53-mediated growth arrest and apoptosis preferentially in MYCN-amplified cells 2) MDMX knockdown increases and p14ARF knockdown decreases the sensitivity of neuroblastoma cell lines to MDM2-p53 antagonists. Methods: The effect of MYCN, MDM2, MDMX and p14ARF was investigated on the response to MDM2-p53 antagonists using siRNA in a panel of 21 neuroblastoma cell lines. Sensitivity was measured by growth inhibition, apoptosis assays including caspase activity and fluorescent activated cell sorting, and the effect on the p53 response measured by Western blotting. Results: Using the SHEP Tet21N MYCN regulatable system, MYCN(-) cells were more resistant to both Nutlin-3 and MI-63 mediated growth inhibition and apoptosis compared to MYCN(+) cells and siRNA mediated knockdown of MYCN in 4 MYCN-amplified cell lines resulted in decreased p53 expression and activation, as well as decreased levels of apoptosis following treatment with MDM2-p53 antagonists. In a panel of cell lines treated with Nutlin-3 and MI-63, the sub-set amplified for MYCN had a significantly lower mean GI50 value and increased caspase 3/7 activity compared to the non-MYCN-amplified group of cell lines, but p53 mutant cell lines were resistant to the antagonists regardless of MYCN status. Knockdown of MDM2 did not alter the apoptotic response to Nutlin-3 or MI-63 but surprisingly, knockdown of MDMX resulted in decreased levels of apoptosis. MDMX expression varied amongst the neuroblastoma cell lines and positively correlated with caspase 3/7 activity following MDM2-p53 antagonist treatment. p14ARF impaired cell lines underwent less apoptosis following MDM2-p53 antagonist treatment and following Nutlin-3 treatment, 3 of 4 p14ARF impaired cell lines underwent a pronounced G1 arrest. p14ARF knockdown alone resulted in decreased caspase 3/7 activity, and following MDM2-p53 antagonist treatment there was decreased caspase 3 cleavage and activity, and decreased PARP cleavage. Conclusions: Amplification or overexpression of MYCN sensitizes neuroblastoma cell lines with wildtype p53 to MDM2-p53 antagonists and these compounds may therefore be particularly effective in treating high risk MYCN-amplified disease. This data also suggests that neuroblastomas with high MDMX expression may be more susceptible to MDM2-p53 antagonist treatment, but that cells with inactivated p14ARF predominantly undergo a G1 arrest which may protect them from apoptosis. MDMX and p14ARF status may therefore be important in addition to MYCN in determining the outcome of neuroblastomas treated with MDM2-p53 antagonists.