Abstract
Cisplatin is an important antitumor agent, but its clinical utility is often limited by multifactorial mechanism of resistance. Loss of tumor suppressor p53 function is a major mechanism that is affected by either mutation in the DNA-binding domain or dysregulation by overexpression of p53 inhibitors MDM2 and MDM4, which destabilize p53 by increasing its proteosomal degradation. In the present study, cisplatin-resistant 2780CP/Cl-16 ovarian tumor cells expressed a heterozygous, temperature-sensitive p53V172F mutation, which reduced p53 half-life by two- to threefold compared with homozygous wild-type (wt) p53 in parental A2780 cells. Although reduced p53 stability in 2780CP/Cl-16 cells was associated with moderate cellular overexpression of MDM2 or MDM4 (<1.5-fold), their binding to p53 was substantially enhanced (five- to eightfold). The analogous cisplatin-resistant 2780CP/Cl-24 cells, which express loss of p53 heterozygosity, retained the p53V172F mutation and high p53-MDM4 binding, but demonstrated lower p53-bound MDM2 that was associated with reduced p53 ubiquitination and enhanced p53 stability. The inference that p53 was unstable as a heteromeric p53wt/p53V172F complex was confirmed in 2780CP/Cl-24 cells transfected with wt p53 or multimer-inhibiting p53L344P mutant, and further supported by normalization of p53 stability in both resistant cell lines grown at the permissive temperature of 32.5 °C. Surprisingly, in 2780CP/Cl-16 and 2780CP/Cl-24 models, cisplatin-induced transactivity of p53 was attenuated at 37 °C, and this correlated with cisplatin resistance. However, downregulation of MDM2 or MDM4 by small interfering RNA in either resistant cell line induced p53 and restored p21 transactivation at 37 °C, as did cisplatin-induced DNA damage at 32.5 °C that coincided with reduced p53-MDM4 binding and cisplatin resistance. These results demonstrate that cisplatin-mediated p53V172F mutation regulates p53 stability at the normothermic temperature, but it is the increased recruitment of MDM4 by the homomeric or heteromeric mutant p53V172F complex that inhibits p53-dependent transactivation. This represents a novel cellular mechanism of p53 inhibition, and, thereby, induction of cisplatin resistance.
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Acknowledgements
The research support from the US Public Health Service Grants CA160687 to ZHS and Support Grant CA16672 to MD Anderson Cancer Center, awarded by the National Cancer Institute, and in part from the Megan McBride Franz Endowed Research Fund, is gratefully acknowledged. We thank Dr Shiaw-Yih Lin (MD Anderson Cancer Center) for the His-Ubiquitin plasmid, and also Dr Guangan He and Michelle Martinez for helpful discussions.
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XX, GL and ZHS designed and/or executed the experiments, and analyzed and interpreted the data. XX and ZHS wrote the initial draft of the manuscript, and GL contributed to revision for the final version.
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Xie, X., Lozano, G. & Siddik, Z. Heterozygous p53V172F mutation in cisplatin-resistant human tumor cells promotes MDM4 recruitment and decreases stability and transactivity of p53. Oncogene 35, 4798–4806 (2016). https://doi.org/10.1038/onc.2016.12
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DOI: https://doi.org/10.1038/onc.2016.12
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