The MDM2 antagonist nutlin-3 sensitizes p53-null neuroblastoma cells to doxorubicin via E2F1 and TAp73

Abstract

Abstract. Neuroblastoma (NB) is a primitive neuroectodermal tumor and the second most common solid tumor in children. NB exhibits heterogeneous behavior and spontaneous regression can occur in patients under 12 months of age. Response to treatment is both age-and stage-specific; however, patients over 1 year of age are generally considered high risk. NB tumors from these patients are often characterized by alterations in p53 expression and murine double minute (MDM2) activity with concomitant resistance to chemotherapy. We evaluated the ability of nutlin-3 to sensitize a p53-null and doxorubicin-resistant NB cell line, LA155N, to doxorubicin. Nutlin-3 treatment upregulated TAp73 and E2F1 protein levels. It potentiated the ability of doxorubicin to block cell proliferation and activate apoptosis and TAp73 knockdown resulted in a reduction of this sensitization. Additionally, PUMA expression was induced by the combination treatment, but reduced by knockdown of either TAp73 or E2F1. We conclude that, following nutlin-3 treatment, TAp73 and E2F1 are released from MDM2 and activated by doxorubicin to induce PUMA and apoptosis. This study addresses p53-independent mechanisms of nutlin-3 action in chemoresistant NB, especially in combination with chemotherapeutics. We believe that this model has strong clinical relevance for chemoresistant and p53 dysfunctional NB.

pRb/E2F-1-mediated caspase-dependent induction of Noxa amplifies the apoptotic effects of the Bcl-2/Bcl-xL inhibitor ABT-737

“…25,26 E2F-1 differs from that of other E2F family members due to its ability to regulate not only cell-cycle progression but also apoptosis as it directly induces the expression of p73, of caspase 3 and 7 and of some pro-apoptotic Bcl-2 family members. [27][28][29][30][31][32][33][34] We show here that E2F-1 is a major contributor of caspase-dependent induction of Noxa in response to ABT-737 treatment. Caspases cleave the E2F-1 regulator pRb in ABT-737-treated cells, giving rise to a p68Rb truncated form, which has a direct role in Noxa and cell death inductions together with E2F-1.…”

“…As treatment with the Mdm2 inhibitor Nutlin-3a was shown to enhance the pRb/E2F-1 pathway, 33,34 we reasoned that it may increase cell sensitivity to ABT-737, provided pRb is present. We found that combination of Nutlin-3a to ABT-737, but not treatment with anyone alone, induced dramatic cell death in the breast cancer cell lines MDA-MB-231 and MDA-MB-468, cells that carry p53 mutations (R280K and R273H, respectively) and constitutively express low levels of pRb (Figures 8a and b).…”

p53-based Cancer Therapy

“…Interestingly several groups have reported the activation of p53 responsive genes such as Puma and p21 and that Nutlin can act in effective synergy with chemotherapeutic drugs to induce apoptosis in p53 defective tumor cells. The effect seems to be linked to the activation of both E2F1 and p73 by nutlin treatment (Ambrosini et al 2007;Kitagawa et al 2008;Lau et al 2008;Peirce and Findley 2009). This in turn is consistent with evidence that Mdm2 binds to both these proteins using the amino-terminal interaction domain that binds to p53 and is blocked by Nutlin.…”

Targeting of p53 and its homolog p73 by protoporphyrin IX

“…Nevertheless, p73/MDM2 interaction is still sufficient to impede p73 transcriptional function. The p53 activator nutlin has been shown to activate p73 in cancer cells [18,19]. Nutlin treatment was shown to induce accumulation of p73 and transcription factor E2F1 with a subsequent induction of the p53-upregulated modulator of apoptosis -PUMA [19].…”

“…The p53 activator nutlin has been shown to activate p73 in cancer cells [18,19]. Nutlin treatment was shown to induce accumulation of p73 and transcription factor E2F1 with a subsequent induction of the p53-upregulated modulator of apoptosis -PUMA [19]. In the recent work Busuttil et al showed that induction of Bim by p73 accounts for the induction of p73-dependent apoptosis in activated T cells with chalcone-inhibited MDM2 [20].…”