Overexpression of GLRX3 Is Associated with Capsular Invasion and Lymph Node Metastasis in Papillary Thyroid Carcinoma

Abstract

Background: Glutaredoxin 3 (GLRX3) is involved in cellular redox homeostasis and iron metabolism and may promote the development and progression of various malignancies. However, the expression profile and clinical significance of GLRX3 in papillary thyroid carcinoma (PTC) remain unclear. This study aimed to investigate GLRX3 expression in PTC and its associations with clinicopathological characteristics.

Materials and methods: This retrospective study included 93 patients with primary PTC who underwent surgery at Zhejiang Cancer Hospital between January 2016 and December 2017. GLRX3 expression in PTC tissues and corresponding adjacent normal thyroid tissues was evaluated by immunohistochemistry and semiquantitatively scored according to staining intensity and the proportion of positive cells. Associations between GLRX3 expression and clinicopathological variables were analyzed using the chi-square test, Fisher’s exact test or McNemar’s test, as appropriate.

Results: Positive GLRX3 staining was predominantly localized in the cytoplasm of PTC cells. High GLRX3 expression was detected in 51 of 93 PTC tissues (54.8%), compared with 3 of 93 matched adjacent normal thyroid tissues (3.2%; P < 0.001). High GLRX3 expression was significantly associated with capsular invasion (P = 0.007) and lymph node metastasis (P = 0.042), but not with sex, age, tumor size, TNM stage, local invasion, distant metastasis, or thyroid-related serological parameters (all P > 0.05).

Discussion: The association of high GLRX3 expression with capsular invasion and lymph node metastasis suggests that GLRX3 may be involved in the aggressive biological behavior of PTC.

Conclusion: GLRX3 is overexpressed in PTC and may serve as a potential marker of aggressive tumor behavior.

Graphical abstract

Keywords

glutaredoxin 3 / papillary thyroid carcinoma / capsular invasion / lymph node metastasis

Introduction

Thyroid cancer is one of the most common primary malignancies of the endocrine system. It comprises papillary thyroid carcinoma (PTC), follicular thyroid carcinoma, medullary thyroid carcinoma, and anaplastic thyroid carcinoma. Among these histological subtypes, PTC is the most common, accounting for approximately 85%–90% of all thyroid cancers^[1,2]. PTC commonly presents as a solid thyroid nodule with irregular margins, although partially cystic lesions may also occur^[3]. Previous studies have advanced our understanding of the molecular pathogenesis of PTC, particularly its genetic and epigenetic alterations, and have provided a molecular basis for targeted therapeutic strategies^[4,5]. Most patients with PTC have a favorable prognosis after conventional treatment, including thyroidectomy, thyroid-stimulating hormone (TSH) suppression and radioactive iodine therapy. However, lymph node metastasis is associated with an increased risk of postoperative structural recurrence^[6] and influences both risk stratification and the extent of cervical lymph node dissection^[7]. Accurate assessment of lymph node metastasis is therefore crucial for prognostic evaluation and clinical decision-making. Further investigation of the molecular mechanisms underlying PTC development and progression, as well as the identification of potential therapeutic targets, is warranted.

Under both physiological and pathological conditions, excessive accumulation of free radicals and their metabolites can disrupt redox homeostasis and induce oxidative stress (OS), leading to tissue or organ damage^[8]. Reactive oxygen species (ROS), the principal class of oxygen-derived free radicals, are intracellularly regulated by the balance between oxidant-producing and antioxidant systems. At physiological levels, ROS are involved in thyroid hormone synthesis, extracellular matrix cross-linking, protein phosphorylation, and the regulation of ion channels and transcription factors^[9]. The thyroid gland is therefore particularly susceptible to OS-induced damage^[8]. Studies have shown that ROS levels are elevated in many malignancies, including thyroid cancer, and may contribute to tumor initiation and progression^[10,11]. Nevertheless, when ROS levels exceed the cellular antioxidant capacity, they can induce cell death^[11,12]. Tumor cells are consequently more dependent on antioxidant systems to neutralize the excessive intracellular ROS generated by their high metabolic activity, thereby supporting tumor progression^[11,12].

Glutaredoxins (GRXs) are important components of the cellular antioxidant defense system and play a central role in maintaining intracellular redox homeostasis^[13]. Using glutathione (GSH) as their principal reducing agent, GRXs limit oxidative damage caused by excessive ROS accumulation by regulating protein S-glutathionylation and deglutathionylation. They also participate in iron metabolism and cellular signal transduction^[14]. Glutaredoxin 3 (GLRX3), an important member of the GRX family, is a glutathione/glutaredoxin-associated redox-regulatory protein^[15,16]. By maintaining cellular antioxidant capacity and limiting excessive ROS accumulation, GLRX3 may support tumor-cell proliferation, survival, migration, and other malignant behaviors^[17,18].

In recent years, GLRX3 has been reported to be highly expressed in breast, colorectal, lung, hepatocellular, and pancreatic cancers, and its expression has been associated with tumor development and progression^[16,19–21]. In breast cancer, high GLRX3 expression is associated with metastasis and poor prognosis. GLRX3 knockdown increases intracellular ROS accumulation and suppresses nuclear factor kappa-B (NF-κB) signaling, thereby reducing tumor-cell proliferation, survival, invasion, and distant metastatic potential^[16]. In pancreatic cancer, GLRX3 knockdown increases intracellular ROS levels in cancer stem cells and inhibits cellular mesenchymal-epithelial transition factor (c-MET)/phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling, impairing sphere formation, proliferation, invasion, tumorigenicity, and distant metastasis in vivo^[21]. Similarly, in nasopharyngeal carcinoma, GLRX3 knockdown induces ROS accumulation, suppresses epidermal growth factor receptor (EGFR)/AKT signaling, and reverses epithelial–mesenchymal transition (EMT), thereby inhibiting cell proliferation, colony formation, migration, invasion, tumorigenicity, and distant metastasis in vivo. Notably, the inhibition of EGFR/AKT signaling appears to be independent of ROS accumulation^[22]. Collectively, these findings indicate that GLRX3 may contribute to tumor progression through antioxidant regulatory mechanisms. However, its role and clinical relevance in PTC remain unclear.

Therefore, the present study aimed to evaluate GLRX3 expression in PTC tissues and adjacent normal thyroid tissues and to analyze its associations with clinicopathological characteristics and tumor progression. These findings may provide preliminary evidence for the potential value of GLRX3 in assessing tumor aggressiveness and guiding further investigation in PTC.

Materials and methods

Patients and samples

A total of 93 patients with primary PTC who underwent surgery at the Department of Head and Neck Surgery, Zhejiang Cancer Hospital, between January 2016 and December 2017 were included in this study. None of the patients had received chemotherapy or radiotherapy before surgery. Tumor tissues and the corresponding adjacent normal thyroid tissues were obtained from the tissue bank of Zhejiang Cancer Hospital and were re-evaluated by two experienced pathologists. Clinicopathological data, including sex, age, maximum tumor diameter, tumor–node–metastasis (TNM) stage, capsular invasion, local invasion, lymph node metastasis, and distant metastasis, were retrospectively collected from the medical records. Thyroid-related serological data were available for 90 patients. Tumors were staged according to the eighth edition of the American Joint Committee on Cancer (AJCC) TNM staging system. This study was approved by the Ethics Committee of Zhejiang Cancer Hospital, and written informed consent was obtained from all patients before surgery.

Tissue microarray construction and immunohistochemistry

Representative tumor areas and corresponding adjacent normal thyroid tissue areas were identified microscopically by pathologists on hematoxylin and eosin-stained sections from all patients. Tissue cores were obtained from the corresponding paraffin-embedded blocks using a tissue microarray (TMA) construction kit (TM-1; Changzhou Ruipin Precision Instrument Co., Ltd. China) and assembled into TMA paraffin blocks. The TMA blocks were incubated at 60 °C for 30 min and subsequently cooled at 21–25 °C to improve tissue-core fixation. Sections of 3 μm in thickness were cut from the TMA blocks for immunohistochemical staining. All sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Antigen retrieval was then performed in 0.01 mol/L citrate buffer. A 3% hydrogen peroxide solution was then used to block the endogenous peroxidase activity. The sections were incubated overnight at 4 °C with a primary anti-GLRX3 antibody (1:100 dilution; Proteintech, 11254-1-AP, China). On the following day, the sections were incubated at room temperature for 50 min with an appropriately diluted horseradish peroxidase-conjugated goat anti-mouse/rabbit secondary antibody (Dako, Glostrup, Denmark). Immunoreactivity was visualized using 3,3′-diaminobenzidine (DAB; Dako), and the nuclei were counterstained with hematoxylin. Finally, the sections were dehydrated through a graded ethanol series, cleared in xylene, and mounted. Negative controls were prepared under each experimental condition by replacing the primary antibody with phosphate-buffered saline.

Evaluation of immunohistochemical staining

GLRX3 immunohistochemical staining was independently evaluated by two pathologists who were blinded to the patients’ clinicopathological data. Discrepant assessments were resolved by joint review and discussion until a consensus was reached. Positive GLRX3 expression was identified primarily by yellow-brown or dark-brown staining in the cytoplasm of tumor cells. Staining intensity was scored as follows: negative (score 0), weak (score 1), moderate (score 2), or strong (score 3). The percentage of positive cells was scored as zero (0%), one (1%–25%), two (26%–50%), three (51%–75%), and four (76%–100%). The final immunohistochemical score was calculated by multiplying the staining-intensity score by the positive-cell proportion score, thereby providing a semiquantitative measure of protein expression. According to the scoring method described by Wen et al.^[23], a final score of < 4 was defined as low GLRX3 expression, whereas a score of ≥ 4 was defined as high GLRX3 expression.

Statistical analysis

Statistical analyses were performed using GraphPad Prism version 8.0.2 and IBM SPSS Statistics version 26.0. Categorical variables were analyzed using the chi-square test, McNemar’s test, or Fisher’s exact test , as appropriate. A two-sided P < 0.05 was considered statistically significant.

Results

Patient characteristics

A total of 93 patients with PTC were included in this study. Of these, 25 (26.9%) were male, 16 (17.2%) were aged ≥ 55 years, 36 (38.7%) had a maximum tumor diameter of ≤ 1 cm, and 87 (93.5%) had stage I–II disease according to the TNM staging system. Capsular invasion was identified in 68 patients (73.1%), local invasion in 22 (23.7%), lymph node metastasis in 44 (47.3%), and distant metastasis in 6 (6.5%).

GLRX3 is aberrantly overexpressed in PTC

To investigate GLRX3 expression in PTC, immunohistochemistry was performed on PTC tissues and the corresponding adjacent normal thyroid tissues. Positive GLRX3 staining was predominantly localized to the cytoplasm of tumor cells and appeared as yellow-brown or dark-brown granular staining. Weak, diffuse staining without clear cytoplasmic localization was considered nonspecific background staining, classified as negative, and excluded from the positive-expression score. Representative immunohistochemical images of GLRX3 expression in PTC tissues and adjacent normal thyroid tissues are shown in Figure 1. According to the immunohistochemical scoring system, among the 93 evaluable pairs of PTC and corresponding adjacent tissues, GLRX3 scores in PTC tissues were 1 in 21 cases, 2 in 11 cases, 3 in 10 cases, 4 in 11 cases, 6 in 35 cases, 8 in 1 case, and 9 in 4 cases. In the adjacent normal thyroid tissues, GLRX3 scores were 1 in 41 cases, 2 in 39 cases, 3 in 10 cases, 4 in 2 cases, and 8 in 1 case. High GLRX3 expression was observed in 51 PTC tissues (54.8%), compared with only 3 corresponding adjacent normal thyroid tissues (3.2%), representing a statistically significant difference (P < 0.001; Table 1).

Associations between GLRX3 expression and clinicopathological characteristics in PTC

The associations between GLRX3 expression and the clinicopathological characteristics of patients with PTC are presented in Table 2. GLRX3 expression was significantly associated with capsular invasion. Among the 93 patients with complete data on capsular invasion, the rate of high GLRX3 expression was 63.2% (43/68) in patients with capsular invasion, compared with 32.0% (8/25) in those without capsular invasion, and the difference was statistically significant (P = 0.007). GLRX3 expression was also significantly associated with lymph node metastasis. The rate of high GLRX3 expression was 65.9% (29/44) in patients with lymph node metastasis, compared with 44.9% (22/49) in those without lymph node metastasis (P = 0.042). The rate of high GLRX3 expression was higher in patients with local invasion than in those without local invasion[72.7% (16/22) vs. 49.3% (35/71)]; however, the difference did not reach statistical significance (P = 0.053). No significant associations were observed between GLRX3 expression and sex (P = 0.891), age (P = 0.669), TNM stage (P = 0.686), maximum tumor diameter (P = 0.456), or distant metastasis (P = 0.216).

Associations between GLRX3 expression and thyroid-related laboratory parameters in PTC

Complete thyroid-related serological data were available for 90 of the 93 patients; the remaining three patients were excluded from this analysis because of incomplete serological records. The associations between GLRX3 expression and thyroid-related serological parameters in patients with PTC were further analyzed, and the results are presented in Table 3. No statistically significant associations were observed between GLRX3 expression and the levels of thyroid peroxidase antibody (TPOAb; P = 0.361), thyroglobulin antibody (TgAb; P = 0.693), thyroid-stimulating hormone (TSH; P = 0.662), or thyroglobulin (Tg; P = 0.894).

Discussion

The development and progression of cancer are closely linked to dysfunction of cellular antioxidant defense systems. Growing evidence suggests that GLRX3 is involved in tumorigenesis and cancer progression^[24]. To our knowledge, the present study is the first to investigate the association between GLRX3 and PTC. GLRX3 expression was assessed by immunohistochemistry in tumor and corresponding adjacent normal thyroid tissues from 93 patients with PTC. GLRX3 was significantly overexpressed in PTC tissues and was associated with capsular invasion and lymph node metastasis. These findings suggest that GLRX3 may contribute to PTC progression.

Previous studies have demonstrated aberrantly high GLRX3 expression in various malignancies. In Ewing sarcoma, high GLRX3 expression has been associated with oncogene-driven tolerance to OS and poor prognosis. GLRX3 knockdown induces a marked accumulation of intracellular ROS and lipid peroxides, thereby triggering ferroptosis and apoptosis and reducing tumor-cell survival and tumorigenic capacity in vivo^[25]. Similarly, in hepatocellular carcinoma, GLRX3 knockdown increases intracellular ROS, disrupts the mitochondrial antioxidant barrier, and suppresses downstream pro-survival signaling, thereby inhibiting cell proliferation and invasion^[20]. Therefore, the overexpression of GLRX3 observed in PTC tissues in the present study is broadly consistent with findings reported in other malignancies. GLRX3 may facilitate PTC-cell survival and invasion by limiting excessive ROS accumulation and enhancing cellular tolerance to OS.

As described above, high GLRX3 expression was significantly associated with capsular invasion and lymph node metastasis in PTC. Capsular invasion reflects the ability of tumor cells to breach local tissue barriers, whereas lymph node metastasis is one of the most common routes of PTC dissemination and may influence recurrence-risk stratification, postoperative surveillance, and decisions regarding the extent of cervical lymph node dissection^[6,7]. A previous meta-analysis showed that patients with papillary thyroid microcarcinoma and capsular invasion had a significantly increased likelihood of lymph node metastasis, suggesting that these features may share an underlying aggressive biological phenotype^[26]. However, capsular invasion is not a prerequisite for lymph node metastasis, as lymph node metastasis may also occur in patients without capsular invasion^[27]. Thus, high GLRX3 expression may be associated with enhanced local invasiveness and regional lymphatic metastatic potential in PTC and may serve as a candidate molecular biomarker for assessing the risks of capsular invasion and lymph node metastasis. Further validation in larger clinical cohorts and functional studies is required to determine whether GLRX3 may provide useful information for surgical decision-making, recurrence-risk stratification, and the development of potential targeted therapies for PTC. Although high GLRX3 expression was more frequently observed in patients with local invasion than in those without local invasion (72.7% vs. 49.3%), the difference did not reach statistical significance (P = 0.053). This observation may warrant further evaluation in larger cohorts to clarify whether GLRX3 expression is also associated with local invasive behavior in PTC.

In addition, no significant associations were observed between GLRX3 expression and sex, age, tumor size, TNM stage, distant metastasis, or serum levels of TPOAb, TgAb, TSH, and Tg. These findings suggest that GLRX3 may be more closely related to capsular invasion and regional lymph node metastasis than to other clinicopathological or thyroid-related serological features.

Conclusion

In conclusion, this study provides the first evidence that GLRX3 is aberrantly overexpressed in PTC tissues and that its expression is associated with capsular invasion and lymph node metastasis. These findings suggest that GLRX3 may be involved in the invasive and metastatic behavior of PTC and provide a basis for further investigation of its role in tumor progression and its potential as a therapeutic target.

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