Adaptation of cancer cells from different entities to the MDM2 inhibitor nutlin-3 results in the emergence of p53-mutated multi-drug-resistant cancer cells

M Michaelis,K Weber,J Cinatl,E Mack,R Breitling,A Von Deimling,J Cinatl,S Barth,B Fehse,N Löschmann,D Speidel,H W Doerr,Y Voges,F Rothweiler,T Stiewe,F Rödel,M Van Rikxoort

doi:10.1038/cddis.2011.129

Abstract

Six p53 wild-type cancer cell lines from infrequently p53-mutated entities (neuroblastoma, rhabdomyosarcoma, and melanoma) were continuously exposed to increasing concentrations of the murine double minute 2 inhibitor nutlin-3, resulting in the emergence of nutlin-3-resistant, p53-mutated sublines displaying a multi-drug resistance phenotype. Only 2 out of 28 sublines adapted to various cytotoxic drugs harboured p53 mutations. Nutlin-3-adapted UKF-NB-3 cells (UKF-NB-3rNutlin10 μM, harbouring a G245C mutation) were also radiation resistant. Analysis of UKF-NB-3 and UKF-NB-3rNutlin10 μM cells by RNA interference experiments and lentiviral transduction of wild-type p53 into p53-mutated UKF-NB-3rNutlin10 μM cells revealed that the loss of p53 function contributes to the multi-drug resistance of UKF-NB-3rNutlin10 μM cells. Bioinformatics PANTHER pathway analysis based on microarray measurements of mRNA abundance indicated a substantial overlap in the signalling pathways differentially regulated between UKF-NB-3rNutlin10 μM and UKF-NB-3 and between UKF-NB-3 and its cisplatin-, doxorubicin-, or vincristine-resistant sublines. Repeated nutlin-3 adaptation of neuroblastoma cells resulted in sublines harbouring various p53 mutations with high frequency. A p53 wild-type single cell-derived UKF-NB-3 clone was adapted to nutlin-3 in independent experiments. Eight out of ten resulting sublines were p53-mutated harbouring six different p53 mutations. This indicates that nutlin-3 induces de novo p53 mutations not initially present in the original cell population. Therefore, nutlin-3-treated cancer patients should be carefully monitored for the emergence of p53-mutated, multi-drug-resistant cells.

Highlights

Eight out of ten resulting sublines were p53-mutated harbouring six different p53 mutations
Continuous exposure to increasing nutlin-3 concentrations for 6–13 passages (Supplementary Table 1) of the neuroblastoma cell lines UKF-NB-3, UKF-NB-2, and UKF-NB-6, the rhabdomyosarcoma cell line UKF-Rhb-1, and the melanoma cell lines Colo-679 and Mel-HO resulted in the formation of p53-mutated sublines (UKF-NB-3rNutlin[10] mM: G245C; UKF-NB-2rNutlin[10] mM: G117W; UKF-NB-6rNutlin[10] mM: K132N, P223L; UKF-Rhb-1rNutlin[10] mM: D281N; Colo-679rNutlin[20] mM: C238F; Mel-HOrNutlin[20] mM: R248Q)
Nutlin-3 inhibits the physical interaction between p53 and its inhibitor murine double minute 2 (MDM2), and activates p53 responses in p53 wildtype cancer cells.[1,2]

Summary

Introduction

Eight out of ten resulting sublines were p53-mutated harbouring six different p53 mutations. Results from the adaptation of a single wild-type p53 cell-derived clone of the neuroblastoma cell line UKF-NB-3 indicate that nutlin-3 induces de novo p53 mutations.

Results

Conclusion