Final Report Summary - P38RADOX (Identification of p38 MAPK partners in the response of endothelium to oxidative stress and ionizing radiation. Impact in cancer radiotherapy.) Projects objectivesCancer is a leading cause of death in Europe and today more than 60% of European cancer patients are treated with radiotherapy. In radiation oncology, current research focuses intensively at delivering more accurate, more efficient and less harmful radiations to increase cytotoxicity towards tumor cells and improve protection of surrounding normal tissues. The microvascular endothelium that irrigates tissues is a major actor of the response to IR and can be considered as a mediator of early and late radiation toxicity to normal tissues. Understanding the biology underlying the cellular response to ionizing radiation (IR) is an on-going challenge to increase the therapeutic ratio (more efficacy, less toxicity) of radiotherapies. Along these lines, signalling events initiated at the plasma membrane are today considered as key issues in tissue response to IR. Therefore, the general objective of the IOF project p38 RadOx is to identify new signalling molecular actor(s) involved in endothelial functional response to oxidative stress induced by ionizing radiation. The p38 MAPK proteins have been identified as critical molecular actors of the membrane initiated-response of the endothelium to IR, through oxidative stress. Better understanding the p38 MAPK pathway in endothelial cells exposed to oxidative stress/IR requires an accurate mapping of this signalling pathway. The p38Radox project is aiming at identifying new partners of p38 MAPK protein in the oxidative stress/IR context in the endothelial compartment. This project benefits from a cellular in vitro approach and from studies on clinical samples of irradiated tissues. Main results and conclusionsBy a proteomic study combined with biochemical and microscopic approaches, we identify a new protein nucleophosmin (NPM), as a new cytosolic partner of p38 MAPK in endothelial cells exposed to massive oxidative stress. This protein, known as a ribonucleoprotein localized in the nucleolus, is involved in several cellular functions mainly in ribosome biosynthesis. Our study now reveals for the first time that this protein forms a heterotrimeric complex with p38 MAPK and the phosphatase PP2a in unstressed endothelial cells. Furthermore, our results describe an unexpected role of p38 MAPK in the endothelial compartment as its acts as a scaffold protein for NPM in the cytosol, maintaining this protein close to the PP2a phosphatase. Oxidative stress and ionizing radiation (15Gy) induces the dephosphorylation of NPM by PP2a, which is then concomitant to NPM translocation from the cytosol to the nucleus. Oxidative-stress and IR provokes DNA breaks and activation of the DNA damage response (DDR) allows cells to repair or not their DNA. Our results show that translocated dephophorylated NPM impacts the DDR, as it impairs detection of the DNA double strand breaks (DSBs), therefore delaying their repair and possibly leading to accumulation of unrepaired DNA damages. Overall, we identify NPM as a new protein of the p38 MAPK pathway in endothelial cells that might contribute to establish genomic instability in cells in response to oxidative stress and ionizing radiations. Identification of NPM in the endothelial compartment in cell culture will be followed by its analysis in human clinical samples of irradiated vascular microenvironment. This translational approach from basic molecular research to validation on clinical human samples is a real strength of this project and collection of samples and development of technical approaches are currently addressed. Impacts The p38 RadOx project identified nucleophosmin NPM as a new molecular actor of the endothelium’s response to oxidative stress and ionizing radiation. We prove that regulation of its localisation/phosphorylation is important for its function upon stress. Therefore, our work identifies the role of NPM in DNA Damage Response (DDR). These results suggest that NPM could participate to the establishment of an endothelial genomic instability, by impairing detection of DNA damages and delaying cellular DNA repair. Genomic instability in endothelial cells is a promoting-factor of dysfunction by blocking cell cycle, inducing senescence or apoptosis. These cellular dysfunctions could ultimately lead to important vascular pathologies (atherosclerosis, ischemia, hypertension). Therefore, NPM’s effect in the nucleus could be an aggravating factor of toxicity in the vascular compartment exposed to oxidative stress and ionizing radiation. Along these lines, NPM could be considered as a potential target to limit endothelial dysfunction and toxicity associated with radiotherapy treatments. Limiting endothelial toxicity and dysfunction induced by oxidative stress/ionizing radiation would help cancer patients treated by radiotherapy to improve their quality of life during and after their treatment.
