Introduction
In recent decades, thyroid cancer has become a very common type of endocrine malignancy whose incidence has rapidly increased worldwide (1). As the most frequent histological type of thyroid cancer, the increased incidence of papillary thyroid carcinoma (PTC) has been pivotal to the increased numbers of patients affected by thyroid cancer (2). More sensitive reliable methods of diagnostic imaging and surveillance have also contributed to the higher detection of PTC (3). Despite the low mortality rate due to PTC, the disease recurrence is high (4). Therefore, exploring the potential molecular mechanisms responsible for carcinogenesis and the development of PTC is imperative.
Similar to other malignancies, thyroid cancer is a complex disease intertwined by multiple genetic and environmental factors (5). Recently, the reduced expression of ribosomal S6 kinase 4 (RSK4) has been implicated in the carcinogenesis of colon, kidney, breast, ovarian and endometrial cancers, suggesting a role as a putative tumor suppressor gene (6-10). RSK4 belongs to the 90-kDa S6 ribosomal kinase family, and its coding gene is located on the X-chromosome (Xq21.1) (9). RSK4 mediates signal transduction downstream of the mitogen-activated protein kinase (MAPK) cascades, serving as a serine/threonine kinase (11). Due to this kinase activity, RSK4 also plays a significant role in the regulation of cellular survival, division and differentiation (9). It has been reported that RSK4 overexpression is capable of inducing a senescence-like phenotype in vitro, while its downregulation can induce the escape from cellular senescence and, consequently, immortalization during the early stages of tumorigenesis (7). PTC is driven by multiple genetic elements, in which the BRAF V600E mutation is the most common oncogenic point mutation, occurring with a prevalence of 45% in PTC (12). BRAF V600E mutation aberrantly activates the MAPK pathway, and plays a crucial role in the carcinogenesis of thyroid cancer (13-15). Furthermore, RSK4 is likely to act directly on or downstream of ERK, therefore inhibiting the MAPK signaling cascade (16).
Epigenetics can be described as a heritable modification of gene function that does not affect the genomic DNA sequence, although ultimately, it leads to a change in phenotype. In the mammalian genome, the methylation of gene promoters is one of the most common epigenetic events (17). As a common feature of tumorigenesis, hypermethylation can block the activity of certain genes which function as tumor suppressors, while demethylation can induce gene reactivation and expression (18). Therefore, the inactivation of gene transcription by promoter methylation may serve as a potential platform for the early diagnosis, risk and prognosis assessment, treatment and management of cancers (19).
The implication of RSK4 methylation in human PTC has not been previously reported, at least to the best of our knowledge. Thus, the present study demonstrates that RSK4 hypermethylation partly contributes to the downregulation of RSK4 expression and is related to the tumor size and lymph node metastasis in PTC. Moreover, the present study investigated the association between RSK4 and the BRAF V600E mutation in PTC. The present study indicates that RSK4 potentially functions as a tumor suppressor and also participates in the carcinogenesis of PTC.
Materials and methods
Patients and tissue samples
A total of 226 paired papillary thyroid carcinoma tissues and corresponding paracancerous counterparts were acquired between January, 2018 and February, 2019, after obtaining informed consents, from patients at the Affiliated Hospital of Qingdao University (Qingdao, China). For patients <18 years old, their written informed consents were provided by their parents. Tissue samples were collected, immediately frozen in liquid nitrogen and stored at -80°C until further analysis. All samples were confirmed by pathological assessment. In this study population, 169 patients were female and 57 were male. The median age at diagnosis was 45 years (range, 17-76 years). Patients with primary PTC were included and those who had received chemotherapy or radiotherapy prior to surgery were excluded. The study design and implementation were approved by the Ethics Committee of the Affiliated Hospital of Qingdao University.
Cells and cell culture
Immortalized thyroid cancer cell lines (TPC-1 and BHT101) and a normal thyroid epithelial cell line (Nthy-ori3-1) were obtained from ExPASy Bioinformatics Resource Portal and Type Culture Collection of the Chinese Academy of Sciences, respectively. TPC-1 and BHT101 cells were cultured in DMEM supplemented with 10% FBS (both from Gibco; Thermo Fisher Scientific, Inc.). Nthy-ori3-1 cells were cultured in F12K medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS. The cells were maintained in incubator at 37°C and 5% CO2. When required, the TPC-1, BHT101 and Nthy-ori3-1 cells were treated with 10 µM 5-Aza-deoxycytidine (5-Aza; Sigma-Aldrich; Merck KGaA) for 5 consecutive days.
Reverse transcription-quantitative RCR (RT-qPCR)
Total RNA was extracted using TRIzol reagent (T9424, Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol. Total RNA (3 µg) was reverse transcribed at 37°C for 15 min and 85°C for 5 sec using the PrimeScript™ RT reagent kit with gDNA Eraser (Code no. RR047, Takara Bio, Inc.). Subsequently, single-stranded cDNAs were subjected to qPCR using TB Green® Premix Ex Taq™ (Code no. RR420, Takara Bio, Inc.) on a LightCycler® 96 Real-Time PCR System (Cobas z480, Roche). The amplification conditions were as follows: 95°C for 30 sec, followed by 30 cycles of 95°C for 5 sec and 60°C for 30 sec. The following gene-specific qPCR primers were used in this study: RSK4 forward, 5'-GAACAG ACATTTCCAGCTAAAAAGG-3' and reverse, 5'-TGCCTG AAGATCCAGCAACTAA-3'; BRAF forward, 5'-TGGGGA ACGGAACTGATTTTTC-3' and reverse, 5'-TTTTGTGGT GACTTGGGGTTG-3'. GAPDH was used as a reference gene (normalizer) and amplified using the following primers: Forward, 5'-TGCACCACCAACTGCTTAGC-3' and reverse, 5'- GGC AT GGACT GT GGT CAT GAG-3'. The relative mRNA expression of RSK4 was analyzed using the 2-AACq method (20).
Western blot analysis
Cell samples were washed twice with PBS and then lysed in Laemmli buffer (2% SDS, 10% glycerol, 0.125M Tris-Cl, pH 6.8). The BCA method was used for protein quantification. The concentrations of separating gel and stacking gel were 10 and 5%. Equal amounts of total protein (20 µg) were resolved by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% skim milk and incubated with respective primary antibodies overnight at 4°C. The membranes were washed 3 times with TBST buffer (containing 0.1% Tween-20) and then incubated with corresponding HRP-conjugated secondary antibody for 1 h at room temperature. The membranes were washed again, 3 times with TBST, prior to protein detection. Specific protein bands were developed by chemiluminescence using a commercial ECL kit (Millipore). The following antibodies were used for western blot analysis: Mouse anti-RSK4 monoclonal antibody (1:500, 27330, Novus Biologicals); rabbit anti-GAPDH polyclonal antibody (1:2,000, E-AB-20059, Elabscience Biotechnology); the secondary antibodies were as follows: Goat anti-rabbit/goat anti-mouse IgG polyclonal antibody (1:10,000, 111-035-003/115-005-003, Jackson ImmunoResearch Laboratories).
DNA extraction and sodium bisulfite modification
Total genomic DNA was extracted from the tissues and cells using a Genomic DNA extraction kit (Aidlab). All procedures followed according to manufacturer's protocol. Genomic DNA samples (3 µg each) was bisulfite-modified and then purified according to the manufacturer's protocol. (Qiagen).
Methylation-specific PCR (MSP)
In total, 134 pairs of specimens of PTC patients were included. Specific primers of RSK4 for the unmethylated (5'-TTTTTATTGATGTTTGGG TGAT-3' and 5'-AACAACAACCACTCAATAATAAC-3') and methylated reactions (5'-TTATTGACGTTTGGGTGAC-3' and 5'-GACAACCGCTCGATAATAAC-3') were utilized. The modified DNA was amplified by PCR using the following conditions: 98°C for 5 min, followed by 35 cycles of 98°C for 30 sec, 56°C for 30 sec and 72°C for 30 sec, and a final 5-min incubation at 72°C. PCR products were isolated by agarose gel electrophoresis.
Bisulfite sequencing (BGS)
In total, 31 pairs of specimens of PTC patients were included. Modified DNA samples were subjected to PCR amplification to further analyze the methylation status of the RSK4 promoter. The primers used for bisulfite genomic sequencing were as follows: Forward, 5'-TTTTAA GTTGGGGAATTTTTATTGA-3' and reverse, 5'-AAAACAACTCTATACTACCTCTCCAAAAAC-3' (21). DNA samples were amplified by PCR using the following conditions: 98°C for 5min, followed by 35 cycles of 98°C for 30 sec, 56°C for 30 sec and 72°C for 30 sec, and a final 5-min incubation at 72°C. PCR products were ligated into the pMD18-T Vector (Takara Bio, Inc.), followed by transformation into competent the Escherichia coli strain, DH5a (Takara Bio, Inc.). At least 6 clones from each ligation reaction were randomly selected. Bacterial solutions were then sequenced by the Beijing Genomics Institute (China). The percentage of methylated DNA for each sample was computed according to the number of methylated CpG dinucleotides/(6×21)×100%.
BRAF V600E mutation analysis
The hot spot region of BRAF V600E mutation was amplified using Premix Taq™ (Takara, Code No. R004). The following PCR primers were used: Forward, 5'-GCTTGCTCTGATAGGAAAATG AG-3' and reverse, 5'-GTAACTCAGCAGCATCTCAGG-3'. Genomic DNA was amplified by PCR using following conditions: 95°C for 3 min, followed by 38 cycles of 95°C for 30 sec, 57°C for 30 sec and 72°C for 45 sec, and a final 10-min incubation at 72°C. Respective PCR products were sequenced by the Beijing Genomics Institute to confirm the BRAF V600E mutation.
Cell transfection
siRNAs targeting BRAF (5'-GCAUCAAUG GAUACCGUUA-3') and control siRNAs (5'-UUCUCCGAA CGUGUCACGU-3') were synthesized by GenPharma. To knockdown BRAF V600E, siRNAs were transfected into the BHT101 cells using HiPerFect Transfection Reagent (Qiagen), according to the manufacturer's protocol. Until transfection, the cells were incubated at 37°C and 5% CO2, and the transfection complexes which had been incubated at room temperature for 10 min were then added to the cells. After 24 h, the cells were harvested and total RNA was extracted.
Cell proliferation assay
The proliferation of the TPC-1 and BHT101 cells was measured using the cell counting kit-8 (MedChemExpress). For this, cells were seeded into 96-well culture plates (2×103 cells/well) and cultured accordingly for 24 h. CCK-8 reagent was added (10 µl/well) at the 0, 24, 48, 72, 96 h followed by incubation for an additional 2 h at 37°C. The absorbance was measured at 450 nm using an enzyme immunoassay analyzer (Gen5 software, BioTek Eliasa).
Statistical analysis
Statistical analysis was performed in silico using SPSS (version 23.0. IBM) and Prism 7 (GraphPad Software Inc.). Data with normal distribution are presented as the means ± SEM. Otherwise, data are expressed as the median with the first and third interquartile range (Q1, Q3). Paired samples from the same patients were compared using the paired Student's t-test. An unpaired t-test or two-tailed Mann Whitney U test was used to analyze the difference between 2 groups. Multiple groups (>3) were compared using one-way analysis of variance ANOVA and the Bonferroni multiple comparisons test. Spearman's correlation analysis was used to analyze the correlation between RSK4 mRNA expression and the methylation rates in PTC tissues. The difference of rate was analyzed using the Chi-square test. P<0.05 was considered to indicate a statistically significant difference.
Results
Reduced RSK4 expression and hypermethylation of its promoter region in PTC
To verify whether the inactivation of RSK4 may contribute to thyroid carcinogenesis, RSK4 expression in PTC tissues and paracancerous counterparts was analyzed by RT-qPCR. The RSK4 mRNA levels were notably reduced in the PTC tissues when compared with their counterparts (n=226, P<0.0001) (Fig. 1A).
In order to assess the possible mechanism of the down- regulation of RSK4 levels in PTC, the methylation status of its gene was examined. A 240-bp region, including 21 CpG sites located in the RSK4 promoter, was analyzed. MSP and BGS analyses were performed to investigate whether a promoter-specific CpG methylation was associated with the decreased RSK4 expression. MSP analysis revealed that the methylation rates of RSK4 in the PTC tissues (93/134, 69.40%) were higher than those in the paracancerous tissues (75/134, 55.97%, P<0.05). RSK4 expression in methylated PTC tissues (n=93) was reduced compared with that in unmethylated PTC tissues (n=41, P<0.05) (Fig. 1C), with no significant difference in expression in the paracancerous tissues (P>0.05). RSK4 expression in methylated PTC tissues was lower than that in methylated paracancerous tissues (n=74, P<0.0001). Moreover, RSK4 expression in unmethylated PTC tissues was also lower than that in unmethylated paracancerous tissues (n=60, P<0.001) (Fig. 1C).
BGS analysis also provided evidence that the methylation rates were higher in PTC tissues (n=31, P<0.05) (Fig. 1D and E). In the majority of CpG sites, the methylation rates of the RSK4 promoter were higher in PTC than in paracancerous tissues (Fig. 1F). In addition, it was found that RSK4 mRNA expression negatively correlated with the methylation rates in PTC tissues (r=-0.37, P<0.05) (Fig. 1G). Taken together, these results indicate that the methylation rates of the RSK4 promoter are increased in PTC, leading to the downregulation of RSK4 expression.
Association between BRAF V600E mutation, and the expression and methylation of RSK4 in PTC
Previous studies have revealed that RSK4 is involved in MAPK signaling transduction and, therefore, it can regulate ERK activation. Thus, the present study investigated whether there was any association between the BRAF V600E mutation with RSK4 expression and methylation in PTC. The chromatograms of DNA sequencing are presented in Fig. 2A, denoting BRAF T1799A and wild-type BRAF. It was found that the RSK4 mRNA levels (PTC relative to the paracancerous tissues) in PTC patients with BRAF V600E mutation (n=124) were significantly lower than those with wild-type BRAF (n=102, P<0.0001, Fig. 2B). Independently of the BRAF mutation status, the RSK4 mRNA levels in PTC tissues were lower than those in paracancerous tissues (Fig. 2C). However, only in patients with BRAF V600E mutation, the methylated rates in the PTC tissues were higher than those in paracancerous tissues, as detected by MSP (73.23 vs. 50.7%, P<0.001) and BGS (P<0.05) (Fig. 2D). Taken together, these findings indicate that BRAF V600E mutation may be one of reasons for the downregulation of RSK4 mRNA levels due to the increasing methylation rates in PTC.
Association between RSK4 methylation levels and the clinicopathological characteristics of patients with PTC
To further explore the role of RSK4 hypermethylation in PTC, 134 (MSP) and 31 (BGS) PTC patients with available information were analyzed. BGS analysis revealed that the methylation rates were higher in tumors with lymph node metastasis (Table I). MSP analysis revealed that the methylation rates in PTC tumors >1.0 cm in diameter (34/41, 82.93%) were higher than those in PTC tumors <1.0 cm in diameter (18/30, 60%) in patients with BRAF V600E mutation (Table II). In addition, the methylation rates were higher in tumors with lymph node metastasis (36/44, 81.82%) compared with those without metastasis (16/27, 59.26%) in patients with BRAF V600E mutation (Table II). PTC patients with Hashimoto thyroiditis were also included to examine whether there was an association between the methylation status of RSK4 and Hashimoto thyroiditis in PTC; it was found that the methylation rate of RSK4 was not related to Hashimoto thyroiditis (Tables I and II). These findings are in accordance with a tumor suppressor role for RSK4 in inhibiting tumor growth and metastasis in PTC.
RSK4 expression associated with BRAF V600E is down- regulated and hypermethylated in thyroid cancer cells
RSK4 levels and the methylation status of its promoter were examined in vitro, using a normal (Nthy-ori3-1) and thyroid cancer cell lines (TPC-1 and BHT101). The results revealed higher methylation rates of the RSK4 promoter in TPC-1 and BHT101 when compared with Nthy-ori3-1 cells (Fig. 3C). By contrast, both the RSK4 mRNA and protein levels were lower in the TPC-1 and BHT101 cells when compared with the Nthy-ori3-1 cells (Fig. 3A and B). As shown in Fig. 3D and E, BRAF expression was significantly downregulated and RSK4 expression was significantly upregulated following the knockdown of BRAF V600E in the BHT101 cells. These findings suggest that RSK4 expression is negatively associated with the methylation status of its promoter and is also influenced by BRAF V600E in thyroid cancer cells. These in vitro data are consistent with the results obtained with the human tissue specimens mentioned above.
RSK4 expression is enhanced by 5-Aza and DNA demethylation inhibits the proliferation of thyroid cancer cells. To further investigate the role of promoter hypermethylation in the regulation of RSK4 expression, the TPC-1, BHT101 and Nthy-ori3-1 cells were treated with the demethylating agent, 5-Aza. The results revealed that the RSK4 mRNA and protein levels were restored in the TPC-1 and BHT101 cells, but not in the Nthy-ori3-1 cells (Fig. 4A and B). Furthermore, the proliferative ability of the TPC-1 and BHT101 cells was significantly suppressed by treatment with 10 µM 5-Aza (Fig. 4C and D). These data suggest that the hypermethylation of the RSK4 promoter can reduce gene expression and, importantly, DNA demethylation can lead to the repression of thyroid cancerous cell proliferation.
Discussion
Papillary thyroid cancer (PTC) accounts for 80-90% of all thyroid cancers and is the most common endocrine malignancy, with a rapidly increasing incidence worldwide (22). Although thyroid cancer has been frequently associated with a relatively indolent progress and a good prognosis, some patients become incurable with notable morbidity and mortality (3,23). In spite of the overall 10-year survival rate (approximately 90%), approximately 10-20% of patients with PTC with stage I or II disease suffer from disease recurrence (24). The molecular pathogenesis of thyroid cancer involves genetic alterations of multiple oncogenes, such as BRAF, TERT and RAS, and tumor suppressor genes, such as PTEN and RASAL1. Their potential association with clinicopathologic aggressiveness and the poor prognosis of thyroid cancer has been partially demonstrated (12,14,25). Advances in the molecular pathogenesis of thyroid cancer have exhibited great prospect toward more effective diagnosis and treatment strategies for this disease (12). Moreover, the understanding of these genetic alterations may further lead to a reduction in unnecessary treatments for indolent PTC, and may also improve the prognosis of patients affected by clinically aggressive cancers (3).
RSK4 is a functional ribosome protein kinase that belongs to the p90 RSK family. Recent studies have demonstrated its apparent significance in tumorigenesis, since a low or no RSK4 expression has been associated with malignant tumors of the colon, kidney, ovary, pancreas, hematological system and endometrium (8,9,26-28). Specifically, it has been demonstrated that RSK4 may drive cellular senescence by modulating Rb and p21, since the upregulation of RSK4 expression can increase p21 levels (6,7,29). These studies have indicated that the downregulation of RSK4 may be an important element in promoting cell transformation and, therefore, may support the notion of the role of RSK4 in regulating senescence as tumor suppressor protein (6-8,16). Similarly, the present study found that RSK4 expression was significantly decreased in the majority of PTC tissues and in a PTC-derived cell line.
Promoter hypermethylation has been recognized as standard mechanism that leads to the transcriptional inactivation of tumor suppressor genes (TSGS) in a variety of cancer types (30). Some studies have indicated that the hypermethyl- ation of the RSK4 promoter can lead to a decrease in its own expression in several malignancies, including breast, endometrial, oral and ovarian cancers. Accordingly, the decreased RSK4 expression has been restored by DNA methyltransferase inhibitors in several cancer cell lines (21,27,30,31). However, no studies to date have reported the methylation status of RSK4 in PTC and paracancerous tissues, at least to the best of our knowledge. Hence, the authors conducted MSP and BGS to finally examine the methylation status of RSK4. Both approaches were able to demonstrate that the methylation rates of RSK4 promoter were consistently higher in PTC than in paracancerous tissues. This increased methylation status negatively correlated with RSK4 expression in PTC tissues. Upon methylation, the RSK4 mRNA levels in PTC were lower than those in paracancerous tissues. Under non-methylating conditions, the RSK4 mRNA levels were less prominently decreased in PTC when compared with those in paracancerous tissues. As stated, it appears that RSK4 methylation may coexist in both PTC and paracancerous tissues, where promoter methylation may be one of the various mechanisms that can lead to a reduction in RSK4 expression. According to the clinicopathological data, this study also demonstrated the association between RSK4 promoter methylation with lymph node metastasis and tumor size, providing evidence that RSK4 methylation may play a role in the development and prognosis of PTC. Indeed, previous studies have indicated that RSK4 can suppress tumor growth, invasiveness and metastatic ability of other cancer cell lines (7,8).
Strikingly, the aberrant RSK4 expression in PTC cells could be reversed by broad-spectrum demethylated drugs, and DNA demethylation inhibited the proliferation of thyroid cancer cells. In other words, the tumor suppressor effect of RSK4, to a certain extent, could be restored by demethylation, suggesting that targeted demethylation drugs may be an attractive option for tumor therapy in the near future.
An extensive number of genetic alterations have been involved in the tumorigenesis of various thyroid cancers. The T1799A transverse point mutation of BRAF is a prototypical example, which leads to the expression of a BRAF V600E mutant protein and contributes to constitutive activation of this serine/threonine kinase. BRAF V600E mutation can activate the MAPK signaling pathway, which is a key feature of PTC (12,32). A previous comprehensive and multicenter study demonstrated a strong association between BRAF V600E with the poor clinicopathological outcomes of PTC, including aggressive pathological characteristics, disease recurrence, loss of radioiodine avidity and unfavorable prognosis (23,33-35). Previous studies have demonstrated that RSK4 can negatively modulate the MAPK pathway at the ERK or the downstream levels, depending on its kinase activity (10,26). The present study analyzed the status of BRAF mutations in 226 PTC tissues and, as a result, it was found that BRAF V600E mutation rates were considerably high. Furthermore, this study indicates that BRAF V600E mutation may influence RSK4 expression, leading to a more significant difference on mRNA levels between PTC tissues and paracancerous tissues. Moreover, it was found that this mutation can influence the methylation status of RSK4, thereby influencing the clinicopathological outcomes of patients with PTC, which reiterates the putative role of RSK4 as a cancer suppressor gene in thyroid tumors.
To conclude, the present study demonstrates that the hypermethylation of CpG islands is one prominent mechanism that can lead the downregulation of RSK4 expression. The hypermethylation of the RSK4 promoter is associated with tumor proliferation and lymph node metastasis. BRAF V600E mutation also has an effect on RSK4 mRNA levels and gene methylation status, supporting the distinctive role of RSK4 in the development, progression and prognosis of PTC.