Clinical significance of oxidative stress markers as angioinvasion and metastasis indicators in papillary thyroid cancer

Angioinvasion remains the important prognostic feature in papillary thyroid cancer (PTC) patients. Literature data indicates several markers that may be associated with oxidative stress and/or angioinvasion. Therefore, we assessed the utility of selected parameters in angioinvasion and metastasis screening in serum of PTC patients. Serum antioxidant capacity (TAC) and sirtuin 3 (SIRT3) levels were decreased (all p < 0.05) and both DNA/RNA oxidative stress damage products (DNA/RNA OSDP) and malondialdehyde (MDA) levels were increased in PTC patients with angioinvasion and metastasis (study group) when compared with PTC patients without these features (all p < 0.01). The highest screening utility in differentiation between angioinvasion and metastasis presence and absence in PTC patients was presented for DNA/RNA OSDP (AUC = 0.71), SIRT3 (AUC = 0.70), and TAC (AUC = 0.67) (all p < 0.05). Our study suggests that peripheral concentration of oxidative stress markers could be useful as angioinvasion and metastasis indicator in PTC patients.

It can be assumed the interplay between oxidative stress, chronic inflammation, and angiogenesis in cancer progression.Thus, our hypothesis is that circulating oxidative stress markers, inflammation and/ or angiogenesis related parameters can be helpful in angioinvasion screening in order to support clinical evaluation in questionable PTC cases.Thus, the aim of this study was to assess the utility of selected parameters in angioinvasion and metastasis screening in serum of PTC patients.

Results
Biochemical characterization.To test the working hypothesis the group of PTC patients was divided into 2 subgroups: PTC patients with both angioinvasion and metastasis (study group) and PTC patients without these features (reference group, considered as very low-risk PTC group according to American Thyroid Association guidelines) 12 (Table1).
The cholesterol (CHOL) and low-density lipoprotein (LDL) concentrations were increased, and high-density lipoprotein (HDL) was decreased in study group when compared with reference group (p < 0.05; p < 0.001; p < 0.05; respectively).Yet, other parameters did not differ among groups (Table 2).

Oxidative stress and PTC angioinvasion and metastasis.
To evaluate the significance of oxidative stress in angioinvasion and metastasis features in PTC, several circulating oxidative stress-related markers were measured in PTC patients.Thus, patients with angioinvasion and metastasis were considered as the study group and patients without these characteristics constituted the reference group.Serum TAC and SIRT3 levels were decreased (all p < 0.05) and both DNA OSDP and MDA levels were increased in study group when compared with reference PTC patients (all p < 0.01).The study and reference groups did not differ in terms of TOC, SIRT1, DNA methylation, FOXO, PRKAA1, PGC1α, p53, NF-κB, and IL-6 (all p > 0.01) (Table 3).

Correlation.
A Spearman correlation analysis was performed to study the relationship between biochemical parameters in total group.The conducted correlation analysis included both groups: the study group, consisting of PTC patients with angioinvasion and metastasis, and the reference group, without the angioinvasion and metastasis, to assess the relationship between studied parameters in PTC patients (Fig. 2).
Considering all the biochemical parameters in PTC patients and their significant associations, strong positive correlation between p53 and NF-κB (r = 0.75; p < 0.001) and between p53 and FOXO (r = 0.79; p < 0.01) was Table 1.The characteristics of PTC patients.F, female; M, male; (m), multifocal; p, pathological; TNM-Cancer tumor-node-metastasis classification (based on the characteristics of primary tumor site (pT)), pT1a-Tumor size ≤ 1 cm in greatest dimension limited to the thyroid, pT1b-Tumor > 1 cm but ≤ 2 cm in greatest dimension, limited to the thyroid, pT2-Tumor size > 2 cm but ≤ 4 cm, limited to the thyroid, pT3/pT4-Tumor size > 4 cm, with gross extrathyroidal extension.

Discussion
The literature data based results assessing the predictive value of angioinvasion in distant metastasis development, especially in well-differentiated thyroid cancer, are discrepant [13][14][15] .This may be due to the inconsistent criteria for angioinvasion detection in endocrine tumors, especially thyroid cancer 13 .Assessment of angioinvasion in primary tumors is currently based upon examination of sections stained with haematoxylin and eosin.Moreover, several vascular markers have been characterized and studied to detect cancer angioinvasion, e.g., a commonly used endothelial marker CD31, platelet-associated protein CD61, and von Willebrand factor (factor VIII-related antigen) [16][17][18][19] .
There is clear evidence for a higher level of oxidative stress, including a compromised antioxidative defense system in cancer 20 .Janion et al. 21showed significantly lower serum MDA and TAS levels and higher TOS levels in the colorectal cancer (CRC) group compared to the control group.Oxidative stress has been also suggested as significant PTC risk factor and the level of ROS generation correlated with cancer aggressiveness 22 .Therefore, potential modulation of oxidative status may influence the cancer progression.It has been hypothesized that oxidative stress markers in thyroid tumors may have diagnostic, prognostic, and therapeutic relevance 22 .To date, little studies have been performed to establish the tools for angioinvasion detection in cancer.Thus, new potential circulating biomarkers indicative of angioinvasion and metastasis are needed.www.nature.com/scientificreports/Accumulating evidence suggest that ROS function as signaling molecules to mediate various growthrelated responses including angiogenesis 23 .It has been observed that oxidative stress stimulates the secretion of angiogenic modulators in cancer cells via hypoxia-dependent and -independent pathways 24 .In our study, the level of serum oxidative stress markers was evaluated to assess the potential screening utility in PTC-related angioinvasion with metastasis.PTC patients with both angioinvasion and metastasis presented decreased serum TAC and SIRT3 and increased DNA OSDP and MDA levels than PTC patients without these features.The results indicate that oxidative stress is involved in angioinvasion development among PTC patients and TAC, Table 3.The oxidative stress markers measurement in PTC patients with angioinvasion and metastasis (study group) versus PTC patients without angioinvasion and metastasis (reference group).Bold indicates that the values given are statistically significant.IL-6, interleukin 6; FOXO, Forkhead box O family member proteins; NF-κB, nuclear factor kappa B; MDA, malondialdehyde; p53, protein 53; SIRT1, Sirtuin 1; SIRT3, Sirtuin 3; TAC, total antioxidant capacity; TOC, total oxidative capacity; PRKAA1 5′-AMP-activated protein kinase catalytic subunit alpha-1; PGC1α, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; MDA, malondialdehyde, DNA/RNA DNA/RNA oxidative stress damage products.www.nature.com/scientificreports/SIRT3, DNA/RNA OSDP, and MDA could be potentially considered as PTC angioinvasion screening tools.TAC measures the peroxyl-scavenging capacity of the extracellular antioxidant system and indirectly reflects the level of oxidative stress.A low serum TAC has been reported to have a strong association with various cancers 25 .Reduced TAC in studied PTC patients when compared with reference PTC patients could reflect the consumption of antioxidants in free radical reactions by the cancer invasion.This suggests that TAC may have a protective role against angioinvasion and metastasis.Based on MDA, lipid peroxidation was significantly increased in PTC patients with angioinvasion and metastasis compared with respective reference group.These results also point to increased oxidative stress in those patients.Furthermore, Maurya et al. 26 demonstrated that pre chemotherapy serum MDA level was significantly higher in patients with primary ocular carcinoma having larger tumor and having lymph node metastasis than those without lymph node metastasis suggestive of its potential significance in cancer progression.
SIRT3, the major deacetylase in mitochondria, plays a crucial role in regulating glucose metabolism, modulating ROS, and limiting the oxidative damage in cellular components 27 .The roles of SIRT3 vary in different types of cancers and they have been thoroughly discussed.In some types of cancer SIRT3 functions as a tumor promoter by lowering ROS levels to maintain cell proliferation, other studies describe SIRT3 as a tumor suppressor, as it can promote apoptosis in cancer cells under stress conditions 28 .SIRT3 plays a crucial role by mediating interactions between mitochondria and intracellular signaling 29 .It has been observed that increased ROS production in SIRT3 null cells contributes to increased HIF1α stabilization leading to the transcriptional activation of multiple metastasis-related pathways 30,31 .Our study suggests that SIRT3 plays a role as angiogenic factor in PTC, however, its involvement in PTC progression is yet to be elucidated.www.nature.com/scientificreports/Interestingly, oxidative damage to nucleic acid has been associated with the development of various diseases including cancer [32][33][34][35] .8-Oxo-7,8-dihydro-2'-deoxyguanosine, 8-oxo-7,8-dihydroguanosine, and 8-hydroxy-2′-deoxyguanosine (8-OHdG) are generated by the oxidative damage to DNA.Increased serum 8-oxodG concentration compared to the control group has been observed in CRC patients 21 .Moreover, it has been demonstrated increased levels of urinary 8-OHdG in CRC patients and patients with tumor metastasis, compared to healthy controls and patients without tumor metastasis, respectively 36 .This line of evidence suggest that peripheral concentration of DNA/RNA OSDP may be useful in noninvasive screening of angioinvasion and metastasis in cancer patients.
Oxidative stress can also activate a variety of transcription factors including NF-κB, FOXO, and p53.P53 is the most extensively studied tumor suppressor deregulated in most human cancers.In turn, NF-κB transcription factors play essential regulatory function in inflammation, cell proliferation, and apoptosis 37 .FOXO proteins are growth factor and stress regulated transcription factors known to cooperate with p53 38 .The reciprocal regulation of these transcription factors can influence several important cancer pathways: tumor cell metabolism, DNA damage, mitochondrial function, and ATP production 39 .Depending on the cellular context, the activation of NF-κB can have different related output to the p53 function.Our study confirmed strong positive correlation between p53 and NF-κB (r = 0.75; p < 0.001) and between p53 and FOXO (r = 0.79; p < 0.01) in PTC patients.FOXO mediates cellular oxidative stress to maintain metabolic stability by promoting transcription of several genes coding for antioxidant proteins 40 .
In turn, PGC1α plays a pivotal role in mitochondrial biogenesis and multiple other pathways (inflammation, endothelial dysfunction, and oxidative stress) 42 .As no difference in serum PRKAA1 and PGC1α concentration was observed between groups, it can be concluded that their peripheral level is not influenced by angioinvasion development as they are produced and exert effects locally.Bauerle et al. demonstrated the key role of NF-κB in angiogenesis and growth of primary and metastatic thyroid cancer 43 .Since, considering these markers, no difference was observed between PTC patients with both angioinvasion and metastasis and PTC patients without these features, the associations need further study to evaluate their role in PTC invasion 44 .Targeting oxidative stress-sensitive pathways and transcription factors offers great promise for cancer prevention and therapy 45 .Nevertheless, the direct roles of ROS and distinct oxidative stress markers in angiogenesis in PTC patients remain to be elucidated.However, serum oxidative status marker levels are good indicators for the systemic oxidant/antioxidant status.
To assess the potential screening utility of the parameters, ROC curves were constructed.DNA/RNA OSDP (AUC = 0.71), SIRT3 (AUC = 0.70), and TAC (AUC = 0.67) have been demonstrated to differentiate between PTC patients with both angioinvasion and metastasis and PTC patients without these features (all p < 0.05).In our regression analysis results, a significant influence of angioinvasion and metastasis on various peripheral parameters, which showed statistical significance (p < 0.05), was observed.Serum parameters such as CHOL, LDL, HDL, TAC, OSDP, SIRT3, and NFκB showed significant association with the presence of both angioinvasion and metastasis in PTC patients.Thus, it can be concluded that the concentration change of these parameters originates from the cancer itselft.In previously conducted studies, we also observed a positive correlation between the concentration of MDA measured among advanced PTC patients and their lipid profile.This finding suggests that individuals with advanced PTC may be at risk of having deregulated lipid levels, which, in turn, is associated with the metastasis process 46,47 .On the other hand, in our analysis, it has been shown the difference in the MDA concentration between groups, however, our regression analysis did not demonstrate the association between angioinvasion and metastasis status and serum MDA level.Therefore, it can be assumed that the change in MDA concentration results from the peripheral oxidative stress rather than the cancer.It can be hypothesized that additional mechanisms involving lipids and MDA are associated with PTC progression, which need further detailed evaluation.Additional investigations on these peripheral metabolic processes will enable to better understand their role and potential clinical significance.Larger studies are also needed to confirm the results and assess the possibility to support of serum oxidative stress markers in challenging PTC screening.
To our knowledge, this is the first study assessing circulating oxidative stress markers as angioinvasion and metastasis indicators in PTC patients.Considering that the parameters were measured in serum, their peripheral concentration results not only from thyroid cancer tissue but also from the imbalance of homeostatic processes.Although the involvement of oxidative stress in PTC angioinvasion and metastasis seems to be important, our hypotheses need to be verified in further molecular studies.
Investigation of thyroid cancer progression from the overview of oxidative stress may be helpful in the understanding of the cancer etiology and may contribute to the development of new approaches to cancer therapy.

Materials and methods
Study design.This research was conducted at the Department of Endocrinology, Diabetology, and Internal Diseases, Medical University of Bialystok, Poland.All patients were diagnosed as having PTC based on clinical laboratory tests and ultrasound imaging, then confirmed by fine needle aspiration biopsy (FNAB), followed by histopathological examination.For this study 80 patients diagnosed with various stages of PTC after total thyroidectomy were enrolled (Table 1).To assess the clinical utility of the selected parameters as indicators of angioinvasion and metastasis, the study group comprised patients with PTC presenting angioinvasion and metastasis (study group), while the reference group consisted of PTC patients without these specific features (reference group).The angioinvasion was identified through post-thyroidectomy histopathological evaluation.To conduct this study, patient recruitment has been ongoing since 2018.
Material and methods.Venous blood (5.5 mL) was obtained and centrifuged, with serum subsequent separation and then frozen at − 80 °C.The procedures were approved by the Local Ethics Committee of the Medical University of Bialystok, Poland, and written informed consent was obtained from each participant (R-I-002/491/2019).
Ethical approval.The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Medical University of Bialystok, Poland (R-I-002/491/2019).
Informed consent.Written informed consent has been obtained from the patients to publish this paper.

Conclusions
Reporting angioinvasion as prognostic factor in PTC patients should be performed to ensure proper management and, therefore, screening tools to for objective evaluation of the process are of great importance.Our study confirmed the screening utility of serum oxidative stress markers as angioinvasion and metastasis indicators in PTC patients.The mechanisms that may connect to the angioinvasion process and oxidative stress in PTC need to be studied in depth.

Figure 1 .
Figure 1.The screening utility of selected oxidative stress markers in angioinvasion and metastasis confirmation in PTC patients based on ROC analysis.

Table 2 .
The biochemical profiles of the study and reference groups.Bold indicates that the values given