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

Abstract

Background: Biomarkers 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.

Methods: We used the isobaric tags for relative and absolute quantitation (iTRAQ) proteomic approach to analyze differences in protein expression levels in ex-vivo irradiated (8 Gy) T lymphocytes from patients with grade ≥ 2 radiation-induced breast fibrosis (grade ≥ 2 bf+) and patients with grade < 2 bf + after curative intent radiotherapy. Patients were selected from two prospective clinical trials (COHORT and PHRC 2005) and were used as discovery and confirmation cohorts.

Results: Among 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 kDa protein; and isocitrate dehydrogenase 2) confirmed AK2 overexpression in 8 Gy-irradiated T lymphocytes from patients with grade ≥ 2 bf + compared with patients with grade < 2 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 ≥ 2 bf + and grade < 2 bf+. Total ROS level, and especially superoxide anion level, increased upon ex-vivo 8 Gy-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 ≥ 2 bf + and grade < 2 bf+).

Conclusion: These 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.

Keywords: AK2; NADPH oxidases; Normal tissue radiotoxicity; Proteomics; Radiation-induced breast fibrosis; Radiosensitivity; Radiotherapy.

Fig. 1

Quantitative Proteomic Analysis. a Schematic overview of the strategy used for the iTRAQ analysis. b Venn diagram showing the distribution of the 1979 identified proteins in each subcellular fraction. c Heat map and patient clustering according to the protein expression profiles. Each row represents one protein; columns represent the T lymphocyte samples of patients with (n = 2) and without (n = 2) grade ≥ 2 bf + after irradiation (8 Gy) or not (0 Gy). In a: eliminate population, change into T lymphocytes

Fig. 2

AK2 is differentially expressed between patients with and without grade ≥ 2 bf+. a Immunoblot analysis of AK2, ANX1, HSPA8 and IDH2 in control (0 Gy) and 8 Gy-irradiated T lymphocytes from the indicated patients. β-actin was used as loading control. b Quantification of the immunoblot presented in A using the ImageJ software. Data are the mean ± SEM; *p < 0.05 (2-tailed Mann-Whitney test). qRT-PCR analysis of AK2 and IDH2 mRNA expression in control (0 Gy) and 8 Gy-irradiated T lymphocytes from the same patients with and without grade ≥ 2 bf+. The graph shows the expression level in irradiated samples relative to non-irradiated samples

Fig. 3

NOX4 mRNA and protein levels are increased in PBMCs after ex-vivo irradiation. a mRNA expression of NOX family members in all patients (n = 20; n = 7 with and n = 13 without grade ≥ 2 bf+) in non-irradiated PBMC samples was analyzed by qRT-PCR. ND, not detected (below threshold). b NOX4 mRNA expression is increased in all patients (n = 20) at 24 h after irradiation (8 Gy) compared with non-irradiated samples (0 Gy) (Wilcoxon matched-pairs signed rank test). c NOX4 protein expression in all patients (n = 20) at 24 h after irradiation (8 Gy) compared with non-irradiated samples (0 Gy) (Wilcoxon matched-pairs signed rank test). d Western blot analysis of NOX4 expression in all 20 patients before (−) and at 24 h after 8 Gy-irradiation (+). β-actin was used as loading control. Data are the mean ± SEM; *p < 0.05 (paired t-test)

Fig. 4

After irradiation (8 Gy), NOX4 mRNA level is significantly increased in PBMCs of patients with grade ≥ 2 bf+. a NOX4 mRNA expression (qRT-PCR analysis) in patients with grade < 2 bf + (n = 13) and with grade ≥ 2 bf + (n = 7) before (circles) and at 24 h after 8 Gy-irradiation (squares). b NOX4 protein expression (western blotting) in patients with grade < 2 bf + (n = 13) and grade ≥ 2 bf + (n = 7) before (circles) and at 24 h after 8 Gy-irradiation (squares) (two-way ANOVA with Bonferroni post-tests). NS: Not Significant; *p < 0.05, by paired t-test. Data are the mean ± SEM