As mentioned previously, mortality due to local recurrence or lymph node metastasis occurs in approximately 10% of papillary thyroid carcinoma (PTC) patients within 10 years [7]. Therefore, there is an urgent need to clarify the pathogenesis of PTC to develop new treatment strategies. The present study found that SIX1 was upregulated in PTC cases overall, with particularly notable upregulation observed in PTC patients exhibiting adverse prognostic features. This highlights the potential significance of SIX1 as a biomarker for identifying high-risk PTC cases and underscores the importance of further research into its role in PTC pathogenesis and potential therapeutic targeting.

SIX1, recognized as a developmental transcription factor crucial in embryonic myogenesis, has been identified to also play a significant role in tumorigenesis. For instance, in cervical intraepithelial or cervical cancer, the expression of SIX1 was markedly higher compared to that in normal cervical tissues [23]. In the current study, SIX1 expression was obviously elevated in PTC compared to ANCT, where almost non-cancerous tissues showed no expression of SIX1. These findings align with previous research indicating the upregulation of SIX1 in PTC while being absent in non-cancerous tissue [16, 17, 24]. Additionally, our findings are consistent with analyses of The Cancer Genome Atlas (TCGA) dataset, which demonstrated higher Six1 mRNA levels in thyroid cancers compared to non-cancerous thyroid tissues [24]. In this regard, functional assays conducted on two thyroid cancer cell lines to examine cell viability and colony formation revealed that SIX1 enhanced PTC proliferation and provided significant protection against apoptosis, whereas knockdown of SIX1 using siRNA inhibited growth rate and colony formation [16, 17, 24]. These results confirm the role of SIX1 in promoting growth in thyroid cancer cells, suggesting its potential as an oncogene in PTC tumorigenesis.

The precise mechanism by which SIX1 mediates tumorigenesis remains unclear [25]. Earlier studies have shown that overexpression of SIX1 facilitates tumorigenesis and proliferation of breast cancer cells by directly activating cyclin A1 transcription [26]. In colorectal and cervical cancers, SIX1 has been implicated in EMT and is described as a mediator of ZEB1 transcription and regulation of TGF-β signaling [23, 27]. More recently, the proliferation of PTC has been linked to the activation of classical STAT3 signaling and the TGF-β/Smad2/3 signaling pathways [16, 17].

In our study, SIX1 was primarily found to be localized in the cytoplasm, a result consistent with the findings of Min and Wei as well as Kong et al. whereas Yang et al. observed nuclear localization of SIX1 expression in their series [16, 17, 20]. High SIX1 expression was detected in 36% of tumors, whereas previous studies have reported a higher positivity ranging from 52 to 63% in PTC tumors [16, 17, 24]. This variance could be attributed to differences in scoring systems, variations in the clone used, or differences in the methodology employed for the study, such as the use of tissue microarrays instead of larger surgical specimens.

Previous literature has consistently associated the upregulation of SIX1 with aggressive tumor characteristics and unfavorable patient prognosis across various cancers [18,19,20]. A recent study linked SIX1’s role in tumor invasiveness to its promotion of glucose metabolism and invasion through the regulation of GLUT3, MMP2, and snail in thyroid cancer [24]. In our study, we found a significant association between high SIX1 expression and aggressive features in PTC, including larger tumor size, multifocality, LNM, tumors with high-grade features, advanced tumor stage, LVI, PNI, and ETE. These results are consistent with other studies [16, 17, 24] that have reported a positive relation between invasiveness and high SIX1 expression. Additionally, our findings align with previous studies utilizing Matrigel invasion assays, which demonstrated that SIX1 upregulation increased invasion in thyroid cancer, while knockdown of SIX1 using siRNA reduced invasive ability in both cell lines. Furthermore, our results are supported by an analysis of TCGA data, which showed positive relations between SIX1 mRNA expression and nodal metastasis [24]. Collectively, these findings suggest that upregulation of SIX1 expression could serve as a biomarker for predicting PTC behavior and clinical outcomes.

Historically, C-PTC and FV-PTC were often regarded as having comparable prognosis and treatment approaches. However, recent research by the TCGA group has revealed distinct differences in clinical outcomes and genetic profiles between these variants. Studies by Henke et al. and Shi et al. [8, 9] examined large series of C-PTC and FV-PTC and found that C-PTC exhibits a higher prevalence of ETE, LNM, disease recurrence, and mortality compared to FV-PTC. They concluded that C-PTC and FV-PTC represent biologically distinct disease entities with different oncogenic drivers and tumor behaviors. In the current study, the clinicopathological parameters appeared comparable between C-PTC and FV-PTC cases. However, this may be attributed to the small sample size of our study.

Little is currently understood about SIX1 expression across various histologic subtypes of PTC. To our knowledge, our study represents the first investigation into the association between immunohistochemical expression of SIX1 and clinicopathological features among both C-PTC and FV-PTC patients. We observed no statistically significant difference in high SIX1 expression between C-PTC and FV-PTC, although there was a slight increase in C-PTC (38.2% versus 30%). This finding contrasts with a recent study that reported a significant difference [16]. The lack of significance in our study may be attributed to the disproportionate number of each histological subtype and differences in the scoring system used.

In the present study, we observed a significant upregulation of SIX1 in C-PTC tumors with larger tumor size, multifocality and high-grade features. Conversely, SIX1 was significantly upregulated in FV-PTC tumors with LVI, LNM, ETE, and advanced tumor stage. Particularly noteworthy in the FV-PTC group, unlike in C-PTC, was the clear association between high SIX1 expression and LNM and ETE, with all LNM patients and nearly all ETE tumors showing high SIX1 expression. It has been suggested that patients with LNM have a higher mortality rate, with incomplete surgical excision being a significant contributing factor to increased mortality in stage I PTC patients [28]. Our findings indicate that SIX1 expression may serve as a useful marker for predicting which patients with clinically node-negative (cN0) FV-PTC may benefit from prophylactic central lymph node dissection.

Previous studies have indicated that locoregional extension to perithyroidal soft tissues and lymph nodes is less common in FV-PTC [8, 9]. In this context, our study may aid in selecting FV-PTC patients who require aggressive management while avoiding overtreatment of the predominantly non-aggressive FV-PTC tumors.

In C-PTC group, the notable association of high SIX1 expression with multifocality holds significant clinical value. This finding suggests that total thyroidectomy, rather than lobectomy or hemithyroidectomy, might be more appropriate for C-PTC patients. This approach could be justified by the potential presence of small foci of disease in the contralateral thyroid lobe, thereby reducing the likelihood of locoregional recurrence.

Moreover, the relation between high SIX1 with tumor size, a key factor in the tumor (T) classification of the AJCC staging system, implies that SIX1 could serve as a predictor of post-treatment recurrence. This suggests that patients with high SIX1 expression may require closer monitoring and more aggressive management strategies to mitigate the risk of recurrence following initial treatment.