Mouse double minute antagonist Nutlin-3a enhances chemotherapy-induced apoptosis in cancer cells with mutant p53 by activating E2F1

Abstract

MDM2 is a critical negative regulator of the p53 tumor suppressor protein. Recently, small-molecule antagonists of MDM2, the Nutlins, have been developed to inhibit the p53-MDM2 interaction and activate p53 signaling. However, half of human cancers have mutated p53 and they are resistant to Nutlin treatment. Here, we report that treatment of the p53-mutant malignant peripheral nerve sheath (MPNST) and p53-null HCT116 cells with cisplatin (Cis) and Nutlin-3a induced a degree of apoptosis that was significantly greater than either drug alone. Nutlin-3a also increased the cytotoxicity of both carboplatin and doxorubicin in a series of p53-mutant human tumor cell lines. In the human dedifferentiated liposarcoma cell line (LS141) and the p53 wild-type HCT116 cells, Nutlin-3a induced downregulation of E2F1 and this effect appeared to be proteasome dependent. In contrast, in MPNST and HCTp53À/À cells, Nutlin-3a inhibited the binding of E2F1 to MDM2 and induced transcriptional activation of free E2F1 in the presence of Cis-induced DNA damage. Downregulation of E2F1 by small interfering RNA significantly decreased the level of apoptosis induced by Cis and Nutlin-3a treatment. Moreover, expression of a dominant-negative form of E2F1 rescued cells from apoptosis, whereas cells overexpressing wildtype E2F1 showed an increase in cell death. This correlated with the induction of the proapoptotic proteins p73a and Noxa, which are both regulated by E2F1. These results indicate that antagonism of MDM2 by Nutlin-3a in cells with mutant p53 enhances chemosensitivity in an E2F1-dependent manner. Nutlin-3a therefore may provide a therapeutic benefit in tumors with mutant p53 provided it is combined with chemotherapy.

HDM2 antagonist Nutlin-3 disrupts p73-HDM2 binding and enhances p73 function

“…This relative difference in Nutlin efficacy based on p53 status is consistent with previous reports (Van Maerken et al, 2006;Ambrosini et al, 2007). Although the inhibition constant (K i ) is reported to be 0.09 mM for Nutlin-3a (Vassilev et al, 2004) and 0.7 mM for Nutlin-3 (Laurie et al, 2006) in p53 wt cells in vitro, numerous studies have reported that 10 mM of Nutlin-3 or Nutlin-3a is needed to disrupt the p53-HDM2 complex and elicit maximal downstream effects in vivo (Vassilev et al, 2004;Laurie et al, 2006;Patton et al, 2006;Tovar et al, 2006;Larusch et al, 2007).…”

“…In contrast, our Nutlin-3 activates p73 LMS Lau et al data demonstrate that Nutlin-3 alone induces p73 protein stabilization as well as enhanced TAp73 transcriptional activity. Thus, the increase in E2F-1 transcriptional activity and p73 protein levels reported by Ambrosini et al (2007) is likely due to the combination treatment with cisplatin chemotherapy and Nutlin-3 since chemotherapies including cisplatin and doxorubicin are known to activate both E2F1 and TAp73 (Irwin, 2004).…”

“…We have previously shown that E2F-1, a transcription factor and an N-terminal HDM2-binding protein, can induce apoptosis independent of p53 by inducing p73 transcription (Irwin et al, 2000). Interestingly, Nutlin-3 has recently been shown to inhibit E2F-1 binding to HDM2, leading to activation of E2F-1 and potentiation of chemotherapy-induced apoptosis in p53-mutant but not wt cells (Ambrosini et al, 2007). However, in our model using Nutlin-3 alone, we did not observe increased p73 transcription or activation of an E2F-1-responsive p73 promoter.…”

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).…”