Evaluation of YB-1 transcriptional factor, DNA glycosylase hNTH1 and human topoisomerase I functions in relation to drug resistance and DNA repair mechanisms

Acquired resistance to anti-cancer therapy is common and is a major clinical issue. Functional DNA repair pathways provide a common mechanism for drug resistance, but it can also result from mutations or reduced expression of the targeted protein. The overexpression or nuclear localisation of the multifunctional Y-box binding protein (YB-1) is considered as a prognostic marker for drug resistance in tumours. YB-1 has several interaction partners in cells; in this study, we have focused on its interaction with the human DNA repair enzyme NTH1 (hNTH1) and human DNA topoisomerase I (hTopoI), two enzymes that have been shown to be stimulated by YB-1. The abundance of the hNTH1/YB-1 complex was shown to increase in cisplatin-resistant tumour cells. Human TopoI is an essential enzyme involved in cellular regulation of DNA supercoiling and is the target of several anti-cancer agents. YB-1 enhances the activity of hTopoI and its sensitivity to hTopoI inhibitor, camptothecin in tumour cells. We have characterised the YB-1/hNTH1 and YB-1/hTopoI complexes in vitro and in vivo using Fluorescence Resonance Energy Transfer (FRET) measurements to identify and develop new strategies for the treatment of drug-resistant tumours. We also designed and optimised an original FRET-based biosensor to screen two medium-sized chemical libraries in order to find potential inhibitors of the hNTH1/YB-1 complex. Several "hits" were identified that significantly reduced the FRET level of our biosensor. For some of these compounds, we have reproduced these results starting from powders, have performed dose-response curves and have validated their actions as inhibitors of the hNTH1/YB-1 interface using alternative binding assays. Taken together, our results demonstrate that YB-1 directly interacts and stimulates a DNA repair and a DNA relaxing enzyme and targeting the YB-1/hNTH1 interface represents an interesting new strategy for the development of anti-cancer drugs.