Functional Analysis of the p53 Pathway in Neuroblastoma Cells Using the Small-Molecule MDM2 Antagonist Nutlin-3

Abstract

Suppression of p53 activity is essential for proliferation and survival of tumor cells. A direct p53-activating compound, nutlin-3, was used in this study, together with p53 mutation analysis, to characterize p53 pathway defects in a set of 34 human neuroblastoma cell lines. We identified 9 cell lines (26%) with a p53 loss-offunction mutation, including 6 missense mutations, 1 nonsense mutation, 1 in-frame deletion, and 1 homozygous deletion of the 3 0 end of the p53 gene. Sensitivity to nutlin-3 was highly predictive of absence of p53 mutation. Signaling pathways downstream of p53 were functionally intact in 23 of 25 cell lines with wild-type p53. Knockdown and overexpression experiments revealed a potentiating effect of p14 ARF expression on the response of neuroblastoma cells to nutlin-3. Our findings shed light on the spectrum of p53 pathway lesions in neuroblastoma cells, indicate that defects in effector molecules downstream of p53 are remarkably rare in neuroblastoma, and identify p14 ARF as a determinant of the outcome of the response to MDM2 inhibition. These insights may prove useful for the clinical translation of evolving strategies aimed at p53 reactivation and for the development of new therapeutic approaches. Mol Cancer Ther; 10(6); 983-93. Ó2011 AACR.

Dual Targeting of Wild-Type and Mutant p53 by Small Molecule RITA Results in the Inhibition of N-Myc and Key Survival Oncogenes and Kills Neuroblastoma Cells In Vivo and In Vitro

“…Nutlin3a has been shown to activate p53-dependent growth suppression in neuroblastoma carrying wild-type (wt) p53 in vitro and in vivo (23,24). Evidence that defects in effector molecules downstream of p53 are remarkably rare in neuroblastoma leads further support to the strategy to restore the function of p53 in neuroblastoma (25). However, recent studies show that treatment with nutlin3a creates a selective pressure for p53 mutations in neuroblastoma and other types of cancer leading to nutlin3a resistance, which in some cases contributes to multidrug resistance (26,27).…”

Mechanism of N-Acylthiourea-mediated Activation of Human Histone Deacetylase 8 (HDAC8) at Molecular and Cellular Levels

“…Yan et al (17) recently demonstrated that the expression of both the wild-type and mutant tumor suppressor protein, p53, in various cancer cell lines are regulated by HDAC8. In this regard, we noted that SH-SY5Y and BE(2)-C cells express wildtype and mutant forms of p53, respectively (38). Given these facts, it appeared plausible that the p53 genotype was responsible for the above noted differential effects in two types of neuroblastoma cells.…”

“…9). The molecular origin of the above selectivity lies in the p53-specific genotype of the above cells (38).…”

“…10). To ascertain whether the above effect is relayed in the downstream cellular process, we evaluated the expression level of p21 (a cell cycle inhibitor), a well characterized target of p53 (38). We observed that the protein expression level of p21 was noticeably enhanced in the TM-2-51-treated SH-SY5Y cells but not in BE(2)-C cells (Fig.…”

Nutlin‐3a selects for cells harbouring TP53 mutations

“…Previous studies indicated that MEFs lacking p19 and p21 are still sensitive to Nutlin‐3a, although loss of p21 appears to hinder the ability of Nutlin‐3a to induce permanent cell cycle arrest 22. Another study showed that loss of p19 potentiated Nutlin‐3a‐induced growth inhibition of neuroblastoma cells 30. Further, MDM2 overexpression reportedly enhances cell sensitivity to Nutlin‐3a 31.…”