• radioinduced thyroid cancer
• DUOX1
• RET/PTC1 rearrangement

Introduction: Differentiated thyroid cancer is the most common endocrine system malignancy, increasing during last 30 years. It is characterized by a lower mortality than other cancers and the survival rate after 5 years is 98%. The most frequent genetic alterations found in these cancers are mutations in BRAF and RAS genes and RET/PTC chromosomal rearrangements. These translocations results by the fusion of RET with unrelated partner genes. Ionizing radiation represents a major risk factor for RET/PTC rearrangements but the molecular mechanisms responsible for this relation are poorly understood. It is known that the thyroid gland is one of the most susceptible organs to the carcinogenic effects of ionizing radiation and about 90% of these cancers are papillary, presenting a RET/PTC chromosomal rearrangement in 70% of cases. A critical role of Reactive Oxygen Species (ROS) in the formation of RET/PTC1 in thyroid cells has been shown. Cells can produce ROS through NADPH oxidases. Recent data show that the NADPH oxidase DUOX1, induced at post-irradiation (post-IR) causes a chronic oxidative stress leading to persistent DNA damage in thyroid cells. The two genes involved in RET/PTC1 are both located in chromosomal fragile sites, which are prone to break under replicative stress. There is now growing evidence that replicative and oxidative stress are intertwined and reinforce each other in driving genomic instability.
Objectives: The hypothesis is that DUOX1-derived ROS may contribute to genomic instability after radiation exposure through generation of replicative stress. The objectives of this study were to investigate the role of DUOX1-derived ROS in a replicative stress which could cause DNA breaks at fragile sites and therefore contribute to the formation of RET/PTC1 rearrangement in irradiated thyroid cells and in papillary thyroid cancer tissues.
Methods: We analyzed, in vitro, the effect of irradiation in terms of oxidative and replicative stress by using the human thyroid cell line (NTHY-ori 3.1). Moreover, we analyzed, by immunohistochemistry, DUOX1 protein expression in papillary thyroid cancer tissues (RET/PTC positive and negative, irradiated and not irradiated) and in normal thyroid tissues and we correlated this data with the epidemiological and clinical-pathological tumoral features.
Results: The data from human thyroid cells showed an increase of reactive oxygen species production and in particular of H2O2 production, in a dose-dependent manner, at day 5 after irradiation and this was related to a persistent increase of oxidative stress due to delayed NADPH oxidase DUOX1 up-regulation, after irradiation. This result was confirmed by a significant increase of DUOX1 mRNA after irradiation, in a dose dependent way. Moreover, we observed that replication stress triggers activation of the ATR kinase pathway, including phosphorylation of the Checkpoint Kinase 1 (Chk1) and the histone H2AX (γH2AX). The analysis of phospho-Chk1 and γH2AX in whole-cell extracts by Western blotting showed a pattern comparable to that of the “double waves profile”. The treatment of cells with Diphenylene Iodonium (DPI), an inhibitor of NADPH oxidase, for 4h at day 4 post-irradiation, significantly decreased the level of γH2AX. We also performed the cell fractioning to evaluate the chromatin-enriched protein fraction after irradiation. In particular, we evaluated the γH2AX expression through western blotting analysis, in chromatin fraction of NTHY-ori 3.1 cells, at 0 Gy and 5 Gy conditions, respectively. The results showed an increase of γH2AX expression after irradiation at dose of 5 Gy in chromatin fraction confirming the presence of DNA damage. The immunofluorescence analysis showed the development of replicative stress after irradiation. In particular, the results showed a great increase in γH2AX, 53BP1 and FANCD2 nuclear characteristic foci after irradiation as well as in merge foci of γH2AX/53BP1 and γH2AX/FANCD2. The radioinduced DNA damage observed several days after irradiation, is materialized in particular by the presence of γH2AX and 53BP1 nuclear foci in G1-phase cells. These foci are likely formed following a replication stress that induces chromosomal damage, which bypass mitosis and are transmitted to daughter cells. Moreover, the increase observed in merge foci between γH2AX and FANCD2, a marker of fragile sites, suggest that the irradiation of NTHY-ori 3.1 cells at dose of 5 Gy promotes the DNA damage in fragile sites. To further investigate the molecular features of replicative stress in irradiated NTHY-ori 3.1 cells, we performed DNA combing to detect the replicative dynamics and in particular the replication speed and fork asymmetry. Importantly, we observed a significative decrease of replication speed at day 4 after irradiation in irradiated NTHY-ori 3.1 cells. The analysis of fork asymmetry between CIdU and IdU tracks did not show significant difference between irradiated and not-irradiated cells. We also performed a ChIP analysis in NTHY-ori 3.1 cells at day 4 after irradiation at dose of 5 Gy using γH2AX antibody to identify which genes presented double-strand breaks, a mandatory step to cause a gene rearrangement. We ran qPCR analysis for RET and CCDC6, the two genes involved in RET/PTC1 rearrangement, and in FRA3B, the most frequently fragile site expressed in the human genome. The preliminary results showed an enrichment of γH2AX (a marker of double-strand breaks), at day 4 after irradiation, in genomic regions mapping between RET and CCDC6 and in FRA3B, indicating that these regions are sensitive to ROS. Moreover, the inhibition of DUOX1 through the shDUOX1, which lead to a decrease of DUOX1 mRNA expression at day 4 post-irradiation in NTHY-ori 3.1 cells, induces a significant reduction of several replicative stress markers expression (i.e. γH2AX, ATR, RPA) at post-irradiation. The immunohistochemistry evaluation of DUOX1 protein expression, performed in 33 papillary thyroid cancer (PTC) tissues (RET/PTC positive and negative, irradiated and not irradiated), showed a moderate-strong staining in the majority of PTC tissues (54.5%) but in only 3% of normal thyroid tissues, used as negative controls with a statistically significant difference in DUOX1 protein expression between PTC tissues and normal thyroid tissues (p<0.0001). We also evaluated the possible correlation between DUOX1 protein expression and clinical-pathological tumoral features. Our results showed that the tumors with tumoral capsule infiltration (p=0.009) and extrathyroidal extension (p=0.009) had a higher expression of DUOX1 than capsulated and intrathyroidal PTC. Moreover, we observed that all 5 PTC tissues with RET/PTC1 rearrangement showed a higher DUOX1 expression than PTC tissues with RET/PTC3 rearrangement (p=0.009) and this data confirm our results in irradiated thyroid cells.
Conclusions: Our study was aimed to understand the role of oxidative stress in a replicative stress in thyroid radio-carcinogenesis that may lead to RET/PTC1 rearrangement generation. Based on our results we can propose that after irradiation, the oxidative burst, through radiolysis of water, activates molecular pathways that may create a persistent oxidative stress through the up-regulation of NADPH oxidase DUOX1. The reactive oxygen species may control the replication fork speed by direct and indirect mechanisms, leading to replicative stress. The untimely replication promotes breaks in fragile sites, in particular in FRA10C and in FRA10G, enabling the formation of RET/PTC1 rearrangement. The inhibition of DUOX1 is accompanied by a significative reduction of the irradiation effects in terms of oxidative and replicative stress and DNA damage in NTHY-ori 3.1 cells. Clinically, the expression of DUOX1 appears stronger in papillary thyroid cancer tissues than in normal thyroid tissues and in particular in presence of RET/PTC1 rearrangement than in presence of RET/PTC3 rearrangement. This strong expression appears to be associated with more aggressive histological features of PTC but it didn’t influence the outcome of the disease.