Due to its DNA binding auto-inhibition, Ets-1 cannot act alone to control its target genes but requires protein interaction partners to bind to target promoters. Given the important role of Ets-1 in tumor invasion, it is crucial to identify these partners and characterize their properties in a relevant model for tumor progression. In this context, we recently identified and validated, in invasive cancer cells, new interaction partners of Ets-1, namely DNA-PK and PARP-1, which are DNA repair enzymes and guardians of the genome integrity.
We characterized the functional interactions between Ets-1 and both DNA repair enzymes. PARP-1 and DNA-PK are both able to modulate Ets-1 transcriptional activity and apply post-translational modification to Ets-1, respectively through PARylation and through phosphorylation, strengthening the functional links between DNA repair enzymes in general and Ets-1. Our recent work highlights that the interactions between Ets-1 and the identified DNA repair enzymes are important for the cancerous cell.
In addition, our very recent results of the mapping of the interaction domains represent very exciting starting points to undertake structural studies in order to understand at the molecular level these interactions and develop inhibitor molecules for these interactions.
With these results in hand, we are currently undertaking the following questions :
Task 1. We will characterize the functional interactions between Ets-1 and the DNA repair enzymes PARP-1 and DNA-PK. We know that both enzymes are able to modulate Ets-1 transcriptional activity. But, what is the impact of these interactions on the cellular genotoxic stress and the DNA repair functions ? We will address this question by dissecting all the steps leading to DNA double-strand breaks which are the most deleterious for the cell survival. This will be done using in-house photonic microscopy facility and expertise. The availability of i) cancer cells over-expressing Ets-1 p27, the dominant-negative form of Ets-1, ii) cancer cells which do not express Ets-1, constitutively or by shRNA and iii) specific pharmacological inhibitors of both enzymes, represent a great advantage for this study. We will follow simultaneously the expression of several Ets-1 target genes involved in tumor progression and the generation of reactive oxygen species (ROS). This detailed study will reveal the links between the transcription factor Ets-1, and the DNA repair enzymes.
Task 2. We will characterize the interaction affinities of the different domains by SPR and initiate structural studies of the complexes, using X-ray crystallography and NMR, which will ultimately reveal the molecular details of these interactions. As the three-dimensional structures of the individual domains are already known, we will also carry out molecular modeling studies. Our team plays a leading role in the CAPRI protein-protein docking experiment, a worldwide collaborative effort aimed at the improvement of computational docking methods. We will use this expertise and employ a molecular docking strategy to identify the binding sites at the atomic level. If the parallel crystallographic studies lead to high resolution crystal structures of one (or several) of the complexes involved, this complex will be used as target in the CAPRI docking experiment. We will use the high detail information from these docking studies to define and develop inhibitor molecules for these interactions. The selected potential inhibitors will be tested in vitro, and the activity of the effective inhibitors of the interactions will be tested on the cancerous phenotypes. Their effect will also be assayed on the transcriptional transactivation of Ets-1 target genes involved in tumor progression and the DNA repair functions of the cancer cells, especially interesting when chemotherapy targeting DNA and radiotherapy are used.
Our results will pave the way to unravel new functions of Ets-1, possibly opening new therapeutic avenues.