Quantitative proteomic analysis reveals AK2 as potential biomarker for late normal tissue radiotoxicity

Abstract

BackgroundBiomarkers for predicting late normal tissue toxicity to radiotherapy are necessary to personalize treatments and to optimize clinical benefit. Many radiogenomic studies have been published on this topic. Conversely, proteomics approaches are not much developed, despite their advantages.MethodsWe used the isobaric tags for relative and absolute quantitation (iTRAQ) proteomic approach to analyze differences in protein expression levels in ex-vivo irradiated (8\u2009Gy) T lymphocytes from patients with grade\u2009≥\u20092 radiation-induced breast fibrosis (grade\u2009≥\u20092 bf+) and patients with grade\u2009<\u20092 bf\u2009+\u2009after curative intent radiotherapy. Patients were selected from two prospective clinical trials (COHORT and PHRC 2005) and were used as discovery and confirmation cohorts.ResultsAmong the 1979 quantified proteins, 23 fulfilled our stringent biological criteria. Immunoblotting analysis of four of these candidate proteins (adenylate kinase 2, AK2; annexin A1; heat shock cognate 71\u2009kDa protein; and isocitrate dehydrogenase 2) confirmed AK2 overexpression in 8\u2009Gy-irradiated T lymphocytes from patients with grade\u2009≥\u20092 bf\u2009+\u2009compared with patients with grade\u2009<\u20092 bf+. As these candidate proteins are involved in oxidative stress regulation, we also evaluated radiation-induced reactive oxygen species (ROS) production in peripheral blood mononuclear cells from patients with grade\u2009≥\u20092 bf\u2009+\u2009and grade\u2009<\u20092 bf+. Total ROS level, and especially superoxide anion level, increased upon ex-vivo 8\u2009Gy-irradiation in all patients. Analysis of NADPH oxidases (NOXs), a major source of superoxide ion in the cell, showed a significant increase of NOX4 mRNA and protein levels after irradiation in both patient groups. Conversely, only NOX4 mRNA level was significantly different between groups (grade\u2009≥\u20092 bf\u2009+\u2009and grade\u2009<\u20092 bf+).ConclusionThese findings identify AK2 as a potential radiosensitivity candidate biomarker. Overall, our proteomic approach highlights the important role of oxidative stress in late radiation-induced toxicity, and paves the way for additional studies on NOXs and superoxide ion metabolism.