Ubiquitin-proteasome genes as targets for modulation of cisplatin sensitivity in fission yeast

Abstract

Background: The ubiquitin(Ub)-proteasome pathway is implicated in the regulation of a variety of cellular functions and plays a major role in stress response in eukaryotic cells, by targeting misfolded and damaged proteins for degradation. In addition, in the presence of DNA damage, the Ub-proteasome system regulates proteins involved in sensing, repairing, and/or tolerating the damage. Antitumor agents such as cisplatin can activate the pathway, but the role of specific pathway components in cell sensitivity/response to the drug is not known. Since platinum compounds represent clinically relevant antitumor agents and a major limitation to their use is the development of drug resistance, there is an urgent need for identifying targets for improving their efficacy.

Results: In the present study, we performed a genome-wide screening for sensitivity to cisplatin using non-essential haploid deletion mutants of the fission yeast Schizosaccharomyces pombe, belonging to a collection of haploid strains constructed through homologous recombination. Using this approach, we identified three Ub-proteasome mutants exhibiting hypersensitivity to cisplatin (ubp16, ubc13 and pmt3) and ten mutants (including ufd2, beta7 20S, rpt6/let1) resistant to the drug. In addition, the importance of lub1 gene emerged from the comparison between the present screening and gene expression profile data previously obtained in fission yeast.

Conclusions: The factors identified in the present study allowed us to highlight most finely the close relationship between the Ub-proteasome system and DNA damage response mechanisms, thus establishing a comprehensive framework of regulators likely relevant also in higher eukaryotes. Our results provide the proof of principle of the involvement of specific genes modulated by cisplatin treatment in cell response to the drug, suggesting their potential role as targets for modulating cisplatin sensitivity. In this regard, the prospective identification of novel targets for modulation of cisplatin sensitivity in an eukaryotic model organism appears particularly intriguing towards the discovery of strategies to overcome cisplatin resistance in human tumors.

Figure 1

Sensitivity of S. pombe deletion mutants to cisplatin. In the bar-chart are reported three cisplatin-sensitive and ten cisplatin-resistant strains with the corresponding human homolog/ortholog genes, when described. IC₅₀ values are the means ± standard deviation of at least three independent experiments. WT, wild-type strains (ED 668: h⁺ ade6-M216/ura4-D18/leu1-32 and ED666: h⁺ ade6-M210/ura4-D18/leu1-32, respectively).

Figure 2

Summary of the relevant findings of the study. The experimental approach used here and the obtained results are illustrated to point out the fact that the Ub-proteasome genes identified in the present study as regulators of cisplatin response are all implicated in controlling DNA damage response.

Figure 3

Cell sensitivity assay. Layout of the standard assay used for screening cell sensitivity of deletion mutants. A growth inhibition assay performed in 96-well microtiter plates was used. After 48 h drug exposure the absorbance at 595 nm was measured.