Snail-1 Overexpression Correlates with Metastatic Phenotype in BRAFV600E Positive Papillary Thyroid Carcinoma

The ability of cancer to metastasize is regulated by various signaling pathways, including transforming growth factor β (TGFβ), also implicated in the upregulation of Snail-1 transcription factor in malignant neoplasms. B-type Raf kinase gene (BRAF)V600E, the most common driving mutation in papillary thyroid carcinoma (PTC), induces epithelial to mesenchymal transition (EMT) in thyroid cancer cells through changes in the Snail-1 level, increasing cell migration and invasion. However, little is known about the mechanism of Snail-1 and BRAFV600E relations in humans. Our study included 61 PTC patients with evaluated BRAFV600E mutation status. A total of 18 of those patients had lymph node metastases—of whom 10 were BRAFV600E positive, and 8 negative. Our findings indicate that the expression of Snail-1, but not TGFβ1, correlates with the metastatic phenotype in PTC. This is the first piece of evidence that the upregulation of Snail-1 corresponds with the presence of BRAFV600E mutation and increased expression of Snail-1 in metastatic PTC samples is dependent on BRAFV600E mutation status.


Introduction
Papillary thyroid carcinoma (PTC) is the most common thyroid cancer accounting for 80-85% of all thyroid cancer cases. Although its incidence is gradually growing, the cancer-related mortality rate is relatively stable, and PTC has a good prognosis with an average 5-year survival rate of above 90% [1]. However, above 50% of PTC case often have performed local lymph node metastasis and approximately 10% of PTC cases represent aggressive metastatic neoplasm with distant metastases to lungs or bones [2]. In addition to an increased rate of primary tumors, there has been also growing incidence of regional and distant PTC metastases, which obviously increases the global need for improving our understanding of pathogenesis process and identifying effective, targeted treatments.
The fundamental mechanism by which epithelial-derived tumor cells may become malignant and obtain an invasive phenotype is the epithelial to mesenchymal transition (EMT). EMT is essential to both physiological developmental processes, as well as metastatic cancer. Numerous studies have demonstrated that this process is aberrantly activated during thyroid cancer development [3,4]. Moreover, it was discovered both in vitro and in vivo that thyroid tumour cells from PTCs and anaplastic cancer (ATCs) may constitutively display an active EMT process when compared to normal to increased Snail-1 expression after malignant transformation, constitutively high ERK activity and resulted in acquisition of a more aggressive cell phenotype [26].
An association has been suggested between BRAF V600E and TGFβ in aggressiveness induction of PTCs. It has been shown in rat thyroid cells overexpressing BRAF V600E that this oncogene stimulates TGFβ secretion, and both proteins exert the same effect on both E-cadherin expression and cell invasion [27]. However, the above studies, based on animal or cell line models (mostly anaplastic thyroid cancer model), may not fully represent the interactions between Snail-1 and BRAF V600E mutation in PTC in vivo.
The aim of our study was to determine whether there is any correlation between Snail-1 and TGFβ1 gene expression and the existence of BRAF V600E mutation in patients with metastatic or non-metastatic PTC. To our knowledge, this study is the first attempt assessing the expression of the above EMT markers and the metastatic potential of PTC in paraffin slides containing human PTC collected during surgery.

Patients
The study group was recruited from PTC patients diagnosed and recruited to the trial during routine follow-up from 2012 at the Endocrinology Clinic of the Holy Cross Cancer Center (Kielce, Poland). Thin-section paraffin-embedded slides of thyroid tissues were prepared by a Holycross Cancer Centre pathologist at the time of diagnosis as decribed earlier [28,29]. One of our pathologists (J. K.) marked the area containing primary tumour foci on one haematoxylin and eosin-stained slide (red line, Figure 1). An association has been suggested between BRAF V600E and TGFβ in aggressiveness induction of PTCs. It has been shown in rat thyroid cells overexpressing BRAF V600E that this oncogene stimulates TGFβ secretion, and both proteins exert the same effect on both E-cadherin expression and cell invasion [27]. However, the above studies, based on animal or cell line models (mostly anaplastic thyroid cancer model), may not fully represent the interactions between Snail-1 and BRAF V600E mutation in PTC in vivo.
The aim of our study was to determine whether there is any correlation between Snail-1 and TGFβ1 gene expression and the existence of BRAF V600E mutation in patients with metastatic or nonmetastatic PTC. To our knowledge, this study is the first attempt assessing the expression of the above EMT markers and the metastatic potential of PTC in paraffin slides containing human PTC collected during surgery.

Patients
The study group was recruited from PTC patients diagnosed and recruited to the trial during routine follow-up from 2012 at the Endocrinology Clinic of the Holy Cross Cancer Center (Kielce, Poland). Thin-section paraffin-embedded slides of thyroid tissues were prepared by a Holycross Cancer Centre pathologist at the time of diagnosis as decribed earlier [28,29]. One of our pathologists (J. K.) marked the area containing primary tumour foci on one haematoxylin and eosin-stained slide (red line, Figure 1). A total of 61 PTC specimens were chosen for further analysis. All the study procedures were approved by the Bioethical Committee at the Holy Cross Medical Chamber, Kielce (no. 2/2013). Patients provided their written consent for molecular tests.The patient characteristics are described in Table 1.  A total of 61 PTC specimens were chosen for further analysis. All the study procedures were approved by the Bioethical Committee at the Holy Cross Medical Chamber, Kielce (no. 2/2013). Patients provided their written consent for molecular tests.The patient characteristics are described in Table 1.
The presence of the V600E mutation in the BRAF gene was evaluated by three genotyping methods: allele-specific amplification PCR (ASA-PCR), qPCR, and Seq as described previously by Kowalik et al. [29]. Within the 61 patients in this study, 30 samples were BRAF V600E positive and 31 BRAF V600E negative. Within 18 patients with lymph node metastasis-10 were BRAF V600E positive, and 8 negative. Controls were obtained from surrounding normal tissue after pathologist supervision.

Molecular Methods
The pathologist marked the area containing PTC tumor cells on a hematoxylin and eosin-stained slide. Then, 4 to 5 unstained slides were deparaffinized by soaking twice in xylene for 15 min, twice in 96% alcohol for 5 min and once in distilled water. Tissue was scraped from thepathologist-selected area with a scalpel and transferred to a test tube for further RNA isolation.
RNA isolation has been performed using a High Pure FFPE RNA Isolation Kit (Roche, Switzerland), according to the manufacturer's protocol. The purity and integrity of total RNA was assessed by an Agilent 2100 Bioanalyzer. The degradation rate of total RNA was determined using RIN values. Only the samples with RIN > 7 were further analyzed. Total RNA was used in the first strand cDNA synthesis with a Maxima First Strand cDNA Synthesis Kit for RT-qPCR with dsDNase (Thermo Scientific, Waltham, MA, USA) according to manufacturer's instructions. RT-qPCR was performed on the ABI PRISM ® 7500 Sequence Detection System (Applied Biosystem, CA, USA) by using TaqMan Gene Expression Assays and TaqMan™ Fast Advanced Master Mix according to the manufacturer's specification. The Assays Identification numbers were: SNAI1-Hs00195591_m1; TGFβ1-Hs00998133_m1; GAPDH Hs99999905_m1. Thermal cycler conditions were as follows: 2 min at 50 • C, hold for 20 s at 95 • C, followed by two-step PCR for 40 cycles of 95 • C for 3 s followed by 60 • C for 30 s. Amplification reactions, in triplicate for each sample, were performed and the results were normalized to the GAPDH gene expression level. An analysis of relative gene expression data was performed, using the 2-∆∆CT method. The calibrator was prepared as mean gene expression values for normal tissue from 12 patients.

Statistics
The statistical analysis was carried out using the Statistica 12 software (StatsoftPolska, Kraków, Poland). A graphical representation of the results was prepared using the SigmaPlot 11 software (Systat Software Inc., San Jose, CA, USA). The results are presented as the mean of three independent experiments ± SD. The normality of the distribution was assessed using the Shapiro-Wilk test. The Student's t-test was used for normally distributed parameters. Chi-square tests and t-test were used in the medical history analysis. In all the analyses, results were considered statistically significant when p < 0.05.

Snail-1, but not TGFβ1 Expression is Correlated with Metastatic Phenotype in PTC
The EMT process may be induced both through transcription factors such as Snail-1 or growth factor TGFβ1. Altered expression of those genes has been observed previously in thyroid cancer cell line models [4,25]. We analyzed the expression of those two genes in 61 FFPE tissues from PTC patients. A total of 18 PTC samples were representing metastases (18 lymph node, with three distant-as described in Table 1)-described later as "Metastatic PTC", and 43 PTC samples had no diagnosed metastases ("Non-metastatic PTC"). RT-qPCR was performed using TaqMan Gene Expression Assays. We observed a statistically significant (p < 0.001), more than 2.5-fold increase in Snail-1 gene expression in metastatic PTC samples as compared to non-metastatic samples ( Figure 2, grey bars). The expression of the TGFβ1 gene in non-metastatic PTC patients was slightly higher, however, no statistical significance for this tendency between the studied groups was noted.
The EMT process may be induced both through transcription factors such as Snail-1 or growth factor TGFβ1. Altered expression of those genes has been observed previously in thyroid cancer cell line models [4,25]. We analyzed the expression of those two genes in 61 FFPE tissues from PTC patients. A total of 18 PTC samples were representing metastases (18 lymph node, with three distantas described in Table 1)-described later as "Metastatic PTC", and 43 PTC samples had no diagnosed metastases ("Non-metastatic PTC"). RT-qPCR was performed using TaqMan Gene Expression Assays. We observed a statistically significant (p < 0.001), more than 2.5-fold increase in Snail-1 gene expression in metastatic PTC samples as compared to non-metastatic samples (Figure 2, grey bars). The expression of the TGFβ1 gene in non-metastatic PTC patients was slightly higher, however, no statistical significance for this tendency between the studied groups was noted.

The Presence of BRAF V600E Mutation Associates with Snail-1 Expression in PTC
As described before, Snail-1 protein expression may be regulated by BRAF V600E mutation in human thyroid cancer cells [25]. Therefore, we divided our studied group into BRAF V600E positive (30 patients) and BRAF V600E negative samples (31 patients). The presence of the V600E mutation in BRAF gene was evaluated previously [14]. Snail-1 mRNA expression was significantly higher (ca. 2-fold) in PTC with BRAF V600E mutation in comparison to BRAF V600E negative samples (p < 0.05) ( Figure 3). However, the expression TGFβ1 did not differ between those two groups. Figure 2. The expression of Snail-1 but not TGFβ1 was correlated with the metastatic phenotype in PTC samples. The gene expression was evaluated by quantitative RT-qPCR in metastatic (n = 18) and non-metastatic (n = 43) PTC groups. The relative mRNA level was normalized to GAPDH. The results are presented as the mean of three independent experiments ± SD. ** p < 0.001.

The Presence of BRAF V600E Mutation Associates with Snail-1 Expression in PTC
As described before, Snail-1 protein expression may be regulated by BRAF V600E mutation in human thyroid cancer cells [25]. Therefore, we divided our studied group into BRAF V600E positive (30 patients) and BRAF V600E negative samples (31 patients). The presence of the V600E mutation in BRAF gene was evaluated previously [14]. Snail-1 mRNA expression was significantly higher (ca. 2-fold) in PTC with BRAF V600E mutation in comparison to BRAF V600E negative samples (p < 0.05) ( Figure 3). However, the expression TGFβ1 did not differ between those two groups.
The EMT process may be induced both through transcription factors such as Snail-1 or growth factor TGFβ1. Altered expression of those genes has been observed previously in thyroid cancer cell line models [4,25]. We analyzed the expression of those two genes in 61 FFPE tissues from PTC patients. A total of 18 PTC samples were representing metastases (18 lymph node, with three distantas described in Table 1)-described later as "Metastatic PTC", and 43 PTC samples had no diagnosed metastases ("Non-metastatic PTC"). RT-qPCR was performed using TaqMan Gene Expression Assays. We observed a statistically significant (p < 0.001), more than 2.5-fold increase in Snail-1 gene expression in metastatic PTC samples as compared to non-metastatic samples (Figure 2, grey bars). The expression of the TGFβ1 gene in non-metastatic PTC patients was slightly higher, however, no statistical significance for this tendency between the studied groups was noted.

The Presence of BRAF V600E Mutation Associates with Snail-1 Expression in PTC
As described before, Snail-1 protein expression may be regulated by BRAF V600E mutation in human thyroid cancer cells [25]. Therefore, we divided our studied group into BRAF V600E positive (30 patients) and BRAF V600E negative samples (31 patients). The presence of the V600E mutation in BRAF gene was evaluated previously [14]. Snail-1 mRNA expression was significantly higher (ca. 2-fold) in PTC with BRAF V600E mutation in comparison to BRAF V600E negative samples (p < 0.05) ( Figure 3). However, the expression TGFβ1 did not differ between those two groups.
Snail-1 expression level was nearly 3-fold higher in metastatic PTC that harbored the BRAF V600E mutation when compared to the metastatic but BRAF V600E negative group (p < 0.05) ( Figure 4A, grey bars). Interestingly, the expression level of the Snail-1 gene in metastatic but BRAF V600E negative PTC samples ( Figure 4A) remained on a similar level as in the non-metastatic PTC patients, regardless of BRAF V600E mutation status ( Figure 4B). Figure 3. The expression of Snail-1 is correlated with the presence of B-type Raf kinase gene (BRAF) V600E mutation in PTC samples. The gene expression was evaluated by quantitative RT-qPCR in BRAFV600E (+) (n = 30) and BRAFV600E (−) (n = 31) PTC groups. The relative mRNA level was normalized to GAPDH. The results are presented as the mean of three independent experiments ± SD. * p < 0.05.
Snail-1 expression level was nearly 3-fold higher in metastatic PTC that harbored the BRAF V600E mutation when compared to the metastatic but BRAF V600E negative group (p < 0.05) ( Figure 4A, grey bars). Interestingly, the expression level of the Snail-1 gene in metastatic but BRAF V600E negative PTC samples ( Figure 4A) remained on a similar level as in the non-metastatic PTC patients, regardless of BRAF V600E mutation status ( Figure 4B). On the other hand, in both metastatic and non-metastatic PTC groups: BRAF V600E (+) or (−) ( Figure 4A,B, white bars), no significant differences in TGFβ1 gene expression levels were found (Student's t-test, p > 0.05 in each case). Table 2 summarizes some vital clinical features of the studied metastatic and non-metastatic PTC patient groups. Statistical analysis showed no significant statistical correlation between BRAF V600E mutation status in PTC tissue and gender, age at diagnosis, or T-classification of the primary tumor. However, it was observed that significantly more samples of non-metastatic PTC tumors with T3-T4 classification harbored the BRAF V600E mutation. Furthermore, it can be observed that more patients with non-metastatic and BRAF V600E negative PTC had been diagnosed as less advanced T1-T2 tumor stage (p = 0.0002). Nevertheless, due to the limited number of patients in the studied groups evaluation of such analysis requires further research. On the other hand, in both metastatic and non-metastatic PTC groups: BRAF V600E (+) or (−) ( Figure 4A,B, white bars), no significant differences in TGFβ1 gene expression levels were found (Student's t-test, p > 0.05 in each case). Table 2 summarizes some vital clinical features of the studied metastatic and non-metastatic PTC patient groups. Statistical analysis showed no significant statistical correlation between BRAF V600E mutation status in PTC tissue and gender, age at diagnosis, or T-classification of the primary tumor. However, it was observed that significantly more samples of non-metastatic PTC tumors with T3-T4 classification harbored the BRAF V600E mutation. Furthermore, it can be observed that more patients with non-metastatic and BRAF V600E negative PTC had been diagnosed as less advanced T1-T2 tumor stage (p = 0.0002). Nevertheless, due to the limited number of patients in the studied groups evaluation of such analysis requires further research.

Discussion
PTC is the most common of all thyroid cancers, with a gradually growing incidence. The majority of PTC cases generally present good prognosis with conventional therapies. Nevertheless, approximately 10% of PTC cases progress to more aggressive forms associated with local invasion, distant metastases and poorer clinical outcome [1,2]. Despite metastasis remaining the key cause of failure in cancer treatment and mortality, its molecular mechanisms remain poorly examined.
Metastatic process is associated with change of phenotype due to the epithelial-mesenchymal transition (EMT), an essential physiological mechanism in embryonic development and tissue repair. However, EMT may also contribute to the progression of disease, such as organ fibrosis or cancer [3]. This process results from the induction of transcription factors that change gene expression, therefore promoting the loss of cell-cell adhesion, modifying cytoskeletal dynamics and leading to a change from epithelial morphology and physiology to the mesenchymal phenotype. EMT can be induced by various signaling pathways, for example mediated by transforming growth factor β (TGFβ), Wnt-β-catenin, Notch, Hedgehog receptor tyrosine kinases (RTK) [4,5].
The role of TGFβ in cancer is complex: this pathway may function both in tumor suppression and in tumor promotion. TGFβ also functions as a tumor suppressor in early tumor development. In a number of human cancers, TGFβ inhibits cell cycle progression, increases apoptosis, and suppresses the expression of growth factors, cytokines, and chemokines. The function of TGFβ as a tumor suppressor or a tumor promoter depends on the context and stage of tumor progression. Its shift to a pro-metastatic role at later tumor stages has been shown to be mediated by various signaling pathways [7,10].
The thyroid gland expresses the TGFβ1 gene mRNA and synthesizes the protein, which under physiologic conditions regulates thyroid growth and function. Interestingly, it was concluded that the alterations in the signaling pathway of TGFβ1 are not the same in tumors from different species [7]. It seems it is not entirely possible to apply the results obtained in animal or cell culture studies to normal or pathological human thyroid tissue. It should be noted that using a single experimental research model usually entails some limitations. For example, employing exclusively tissue material collected from patients, may lead to a somehow less comprehensive scientific reasoning.
The studies of the expression of TGFβ1 performed before in human PTC revealed that, when compared to the central areas, the peripheral invasive areas of the tumors had increased expression of TGFβ1, NFkβ, and CDC42 as well as showing a decrease in cell-cell adhesion proteins. Moreover, these regions presented also an overexpression of vimentin, a characteristic marker of EMT. The authors concluded that these changes might be related to increased tumor invasiveness and suggest an important role of TGFβ as inductor of EMT and metastasis [5,7]. Other studies of Brace et al. have shown that TGFβ1, but not TGFβ2, is significantly increased in PTC compared to benign thyroid nodules and therefore may serve as a potential diagnostic marker [30].
However, in our studies, no statistically significant differences were found in TGFβ1 gene expression in BRAF V600E (+) and BRAF V600E (−) PTC groups, as well as in PTC metastatic samples with known BRAFV600E mutation status. Possibly, this may result from the fact that TGFβ has been found overexpressed locally in the invasive front of cancer cells, also in different cancer types, for example in the breast cancer [31]. Moreover, in ovarian cancer Wang et al. have demonstrated that the expression of TGF-β1 is not associated with lymphatic metastasis [32]. Moreover, it has been discovered that BRAF V600E increases exclusively TGFβ1 secretion through the MEK/ERK pathway in thyroid tumour cells [27]. The observations of Riesco-Eizaguirre et al. have led to the concept that the action of TGFβ in PTC is very important locally as a modulator of the tumor microenvironment, however, the total TGFβ1 expression may not be altered [33].
It has been widely described that the TGFβ cascade also induces the expression of Snail-1-a zinc finger transcription factor, the main E-cadherin repressor and one of the most potent EMT inducers [34]. Snail-1 was found to be overexpressed in the invasive fronts of several human tumors derived from epithelial cells [4,14] and proved to play a key role in PTC, as well. Although Snail-1 is not expressed in normal thyroid tissue, histopathological analyses have proved that the expression of Snail-1 protein was mainly located in the cytoplasm and nuclei of PTC cells [19]. The overexpression of this gene and the upregulated protein level have been discovered in numerous cancer types such as ovarian and breast carcinomas, melanomas, oral squamous carcinomas, human thyroid carcinomas and their metastases. In the context of thyroid cancers, the effects of Snail-1 interactions have not been thoroughly studied or understood. It has been suggested that Snail-1 expression, together with TGFβ1, is associated with lymph node metastasis in PTC and may be a potential biomarker of tumor diagnosis and prognosis in PTC [8].
Interestingly, Baquero et al. have discovered that Snail-1 expression may also be regulated by BRAF V600E -the most common mutation found in PTCs [25]. In the thyroid carcinoma cell model, BRAF V600E induced EMT via changes in Snail-1 and E-cadherin protein expression levels, which in turn increased the migration and invasiveness of these cells. Moreover, inhibition of BRAF V600E significantly decreased invasion of thyroid cancer cells, tumor volume and metastases in a mouse model of anaplastic thyroid carcinoma [25,27].
In our studies, it was observed that BRAF V600E mutation was present in significantly more cases of non-metastatic aggressive tumors (with T3-T4 classification). However, due to the limited number of patients in the studied groups, such statistics should be evaluated with some perspective.
Most clinical research has demonstrated an association between BRAF V600E mutation status, aggressive clinicopathologic characteristics of PTC and high tumor recurrence, although the results remain controversial [24]. Contradictory results from other groups have stressed that, although BRAF V600E mutation may show very high prevalence of the BRAF mutation in PTC in the population, the presence of the mutation itself was not statistically associated with a metastasis, patient age, completeness of resection, local invasion or tumor size score. Although BRAF V600E analysis may have some value in distinguishing malignant and benign thyroid tumors, it is currently believed that alone it is not a good predictor of more the aggressive clinical course of PTC [28].
Therefore, we have aimed to evaluate if Snail-1 and TGFβ1 genes expression correlate with one another in human PTC tissue samples or whether they are related to BRAF V600E mutation status or LNM tumor staging. We demonstrate that Snail-1 gene expression is significantly increased in metastatic PTC patients group (based on LNM scale) in comparison to non-metastatic samples. Moreover, we show that Snail-1 elevated expression also correlates with BRAF V600E positive status. Our results clearly correspond with the data obtained by Baquero et al. on the cellular model [25]. On the contrary, Mitchell et al., utilizing immunohistochemical techniques, suggested that Snail-1 protein expression did not correlate with BRAF status and E-cadherin expression in PTC [35]. Such discrepancies may result from different molecular techniques, or the relatively small number of studied patient samples. It must be also emphasized that Snail-1 protein is relatively unstable, and in the cytoplasm, it may be prone to proteasomal degradation, with a half-life of only twenty-five minutes. GSK3β is a Snail kinase can bind to and phosphorylate this transcription factor, facilitating its degradation. Phosphorylation determines Snail-1 s subcellular location, as GSK-3β-mediated phosphorylation induces Snail-1 export to the cytoplasm [9,36]. Therefore, every study concerning Snail-1 protein expression should also consider its localization.
Interestingly, for the first time using human PTC tissue model, our findings reveal that Snail-1 gene expression was significantly higher exclusively in metastatic PTC that harbored the BRAF V600E mutation, but not in metastatic BRAF V600E -negative PTC tissue. In contrast, we did not observe such differences in Snail-1 gene expression in non-metastatic PTC patients, regardless of BRAF V600E mutation status. This may suggest that in cells altered by metastatic processes, such as EMT, BRAFV 600E mutation facilitates the mechanism of Snail-1 overexpression. It has previously been proposed that BRAF V600E can increase Snail-1 expression both at transcriptional level and via GSK3β inhibition [27]. However, the mechanism of transcription regulation in the presence of BRAF mutation remains to be further elucidated.
Despite some limitations of our study resulting from the relatively small sample size due to the selection of metastasizing PTC cases, caused by only one mutation of individual gene (BRAF V600E ), the presented data confirm that BRAF V600E participates in mechanisms involved in the increase in Snail-1 expression in metastatic PTC. Possibly, the interplay between BRAF V600E and Snail-1 may play a key role in the thyroid gland neoplastic transformation and cancer cell invasiveness. The further elucidation of the mutual interactions between Snail-1 and BRAF V600E mutation in PTC in vivo may shed new light on the issue of metastatic thyroid cancer development. Better understanding of its complexity would help to identify specific targets for modern molecular therapies and develop more accurate diagnostic strategies.

Conflicts of Interest:
The authors declare no conflict of interest.

PTC
Papillary thyroid carcinoma AT Anaplastic thyroid carcinoma EMT Epithelial to mesenchymal transition TGF Transformation growth factor CSC Cancer stem cells LNM Lymph node metastases MAPK Mitogen-activated protein kinase TUBB3 Tubulin -β3, RTK-Receptor tyrosine kinases