Changes in antioxidant status and DNA repair capacity are corroborated with molecular alterations in malignant thyroid tissue of patients with papillary thyroid cancer

Abstract

Introduction: Papillary thyroid cancer (PTC) accounts for approximately 80% of all thyroid cancer cases. The mechanism of PTC tumourigenesis is not fully understood, but oxidative imbalance is thought to play a role. To gain further insight, this study evaluated antioxidant status, DNA repair capacity and genetic alterations in individuals diagnosed with benign thyroid lesion in one lobe (BTG) and PTC lesion in another. Methods: Individuals with coexisting BTG and PTC lesions in their thyroid lobes were included in this study. Reactive oxygen species (ROS) level, ABTS radical scavenging activity, ferric reducing antioxidant capacity, glutathione peroxidase and superoxide dismutase activities were measured in the thyroid tissue lysate. The expression of selected genes and proteins associated with oxidative stress defence and DNA repair were analysed through quantitative real-time PCR and Western blotting. Molecular alterations in genomic DNA were analysed through whole-exome sequencing and the potentially pathogenic driver genes filtered through Cancer-Related Analysis of Variants Toolkit (CRAVAT) analysis were subjected to pathway enrichment analysis using Metascape. Results: Significantly higher ROS level was detected in the PTC compared to the BTG lesions. The PTC lesions had significantly higher expression of GPX1, SOD2 and OGG1 but significantly lower expression of CAT and PRDX1 genes than the BTG lesions. Pathway enrichment analysis identified "regulation of MAPK cascade," "positive regulation of ERK1 and ERK2 cascade" and "negative regulation of reactive oxygen species metabolic process" to be significantly enriched in the PTC lesions only. Four pathogenic genetic variants were identified in the PTC lesions; BRAF ^V600E, MAP2K7-rs2145142862, BCR-rs372013175 and CD24 NM_001291737.1:p.Gln23fs while MAP3K9 and G6PD were among 11 genes that were mutated in both BTG and PTC lesions. Conclusion: Our findings provided further insight into the connection between oxidative stress, DNA damage, and genetic changes associated with BTG-to-PTC transformation. The increased oxidative DNA damage due to the heightened ROS levels could have heralded the BTG-to-PTC transformation, potentially through mutations in the genes involved in the MAPK signalling pathway and stress-activated MAPK/JNK cascade. Further in-vitro functional analyses and studies involving a larger sample size would need to be carried out to validate the findings from this pilot study.

Keywords: benign thyroid goitre; oxidative DNA damage and repair; papillary thyroid cancer; reactive oxygen species; signalling pathways; whole-exome sequencing.

FIGURE 1

Oxidative stress and antioxidant response in tissue lysate samples from benign and malignant lesion. (A) ROS detection analysis; (B) ABTS radical scavenging activity; (C) ferric reducing antioxidant power (FRAP) assay; (D) glutathione peroxidase activity; (E) superoxide dismutase (SOD) activity. * indicates significant difference (p < 0.05) between benign and malignant lesions.

FIGURE 2

Gene expression analysis of OGG1, CAT, GPX1, SOD1, SOD2, PRDX1 and PRDX6 through qRT-PCR. * indicates significant difference (p < 0.05) between the gene expression in benign and malignant lesions.

FIGURE 3

Protein expression analysis of OGG1 and PRDX6 in the BTG and PTC lesions from six PTC patients through Western blotting. (A) Western blot gel image of β-actin, OGG1 and PRDX6 in BTG lesions and PTC lesions of the patients. (B) The relative expression of OGG1 and PRDX6 in PTC lesions against BTG lesions after normalisation to the β-actin expression. * indicates significant difference (p < 0.05) between the gene expression in benign and malignant lesions.

FIGURE 4

Venn diagram illustration of the genes contained filtered variants after CRAVAT analysis in BTG and PTC lesions. Nonsynonymous SNVs with p-values <0.05 in both CHASM and VEST algorithms were prioritised, whereby InDels and splice site variants with p-values <0.05 in VEST algorithm were prioritised. The genes with these retained variants were then used for pathway and process enrichment analysis.

FIGURE 5

Gene enrichment analysis showing the top enriched pathways based on the GO ontology database. (A) The top enriched GO parental terms in benign and malignant lesions. The differentially enriched GO parental terms between benign and malignant lesions are red line bordered. (B) The top enriched GO terms within the four differentially enriched parental terms. (C) The genes involved in GO:0043408, GO:0070374 and GO:2000378. The three ontology terms and their closely related terms are indicated with arrows. * indicates the CRAVAT stratified driver genes that are presented in PTC lesions only. Ontology term with p-value <0.01 (LogP < −2) was considered as significant. (D) Venn diagram illustrates the distribution of the potential driver genes stratified by CRAVAT that involve in MAPK signalling cascade and its regulation, as well as regulation of ROS metabolic process, between BTG lesions and PTC lesions.

FIGURE 6

Detection of BRAF-rs113488022 (BRAF ^V600E) through Sanger sequencing. The position of the c.1799T>A mutation is indicated by the arrow: (A) mutant BRAF ^V600E and (B) wildtype.