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Introduction

Thyroid cancer is the most common endocrine malignancy, which affected ~63,000 persons in the USA, in 2014. Studies between 1975 and 2009 revealed an annual increase in incidence rate from 4.9 to 14.3 per 100,000 (1,2). Thyroid cancer can be categorized according to histopathological findings. Papillary thyroid carcinoma (PTC) is the most prevalent subtype that accounts for ~70–80% of all thyroid cancer cases. One report predicted that the incidence of PTC will double in the USA by 2019 (3,4). The current protocol for treating PTC involve the combination of thyroidectomy and radioiodine therapy. However, these are often not curative for cases that exhibit aggressive clinical characteristics. Hence, a better understanding in molecular pathogenesis and mechanism in PTC would enable us to develop more effective treatment targets and strategies.

WNT and its signaling pathways play a crucial role in human organogenesis (5). In human, WNT genes encode secretory signaling glycoproteins that are cysteine rich and are 350–400 amino acids in length (6). Its corresponding signaling pathways are classified according to the involvement of β-catenin: the canonical (WNT-β-catenin pathway) (7) and the non-canonical pathways (WNT/planar cell polarity (PCP) or WNT/Ca2+ pathway) (8).

In the canonical pathway, WNT ligands bind to Frizzled receptors that induce phosphoprotein Dishevelled for activation. This activation would inhibit β-catenin phosphorylation by glycogen synthase kinase-3β-adenomatous polyposis coli-axin complex. Consequently, β-catenin accumulates in the cytoplasm and eventually translocate into the nucleus, where it acts as a transcriptional coactivator alongside the T-cell factor and lymphoid enhancing factor (TCF/LEF). They form complexes that initiate the expression of downstream genes such as cyclin D1 and c-myc. Such activation seems to play an important role in cell proliferation, differentiation, cell-cell adhesion and cell migration (6,9,10). Hence, any disruption in this pathway would contribute to tumorigenesis.

WNT10A is one of the nineteen WNT signaling glycoproteins and it is the focus of this study. The WNT10A genes are encoded on human chromosome 17q21 (11). Previous studies indicated any aberration of WNT10A in human can induce defects in tooth morphogenesis, dentinogenesis, odontoblast differentiation, hair follicle development, papillae of the tongue and sweat gland, nail formation, and regeneration of the epidermis (12–14). In addition, WNT10A mutation was demonstrated to play key roles in carcinoma of esophageal, gastric, kidney and colorectal as well as endometrioid carcinoma.

Further studies into cellular level revealed that mutation promotes tumor cell proliferation and migration, which may be linked to self-renewal of a subset of ESCC cells in esophageal squamous cell carcinoma by regulating β-catenin (15–18). In concurrence, the knockdown of WNT10A suppressed cell proliferation. It also induces S-phase cell cycle arrest in mouse embryonic palatal mesenchymal (MEPM) cells through WNT/β-catenin signaling pathway (19). However, the association of WNT ligands and its pathway in the pathogenesis of PTC and progression have not yet been determined. In this study, the WNT10A/β-catenin pathway was demonstrated to promote and play a role in the PTC development.

Materials and methods

Specimens

A total of 35 cases of primary papillary thyroid cancer and 35 cases of adjacent normal tissues were collected as fresh frozen tissues from Shandong Provincial Hospital. All samples were collected under the approved guidelines of Shandong Provincial Hospital's institutional review board. The protocols were reviewed and approved by the ethic committee of Shandong University (Jinan, Shandong, China).

Microarray analysis

The microarray chip consisted of 27,326 different human cDNAs (Angilent, Wilmington, DE, USA), in which house-keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal control. The cDNAs from 5 cases of papillary thyroid cancer were labeled with Cy5, and the cDNAs from 5 cases of adjacent normal tissues were labeled with Cy3. The labeled cDNAs were hybridized with microarray chip under standard conditions according to the manufacturer's instructions. The data were analyzed by Molecule annotation system 3.0.

Cell culture and transfection

Thyroid cancer cell lines GLAG-66 was used in this study. All thyroid cancer cell lines were maintained in our laboratory. GLAG-66 was maintained in Nutrient mixture F-12 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (2%) at 37°C with 5% CO2, as described previously (20). Cells were passaged 1:3 until 80% confluence was reached in a 75-cm2 culture flask (Nest Biotechnology, Shanghai, China). The ORF human WNT10A cDNA expression plasmid and LEF1 cDNA expression plasmid were purchased from Biosune Co. (Shanghai, China). FuGENE HD transfection reagent (Roche Applied Science, Basel, Switzerland) was used for transfection. All transfections were performed according to the manufacturer's instructions.

SiRNA interference

Chemical modified Stealth siRNA (chemical modified stealth siRNA) targeting WNT10AsiRNA and LEF1siRNA were from RiboBio Co., Ltd. (Guangzhou, Guangdong, China). The sequence for WNT10A siRNA was 5′-CCACGAATGCCAACACCAA-3′. The sequence for LEF1 siRNA was 5′-GCTACATATGCAGCTTTAT-3′. Cells were transfected with siRNA by the Lipofectamine 3000 method (Life Technologies, CA, USA).

RNA isolation and quantitative real-time PCR

Total cellular or tissue RNA was extracted with TRIzol (Life Technologies) according to the manufacturer's instructions. First-strand cDNA was synthesized from 1 µg total cellular or tissue RNA using PrimeScript™ RT Master Mix (Takara) with random primers. Then cDNA was amplified for quantitative real-time PCR, and the specific primers used were as follows: for WNT10A, forward, 5′-TCCCATCTTCAGCAGAGGTTTC-3′ and reverse, 5′-CACTGCCTGCCTCCCAACT-3′; for β-actin, forward, 5′-AGTTGCGTTACACCCTTTCTTG-3′ and reverse, 5′-CACCTTCACCGTTCCAGTTTT-3′; for LEF1, forward, 5′-AGAGGAAGGCGATTTAGC-3′ and reverse, 5′-ACCACGGGCACTTTATTT-3′; for CTNNB1, forward, 5′-GCAGCAACAGTCTTA CCT-3′ and reverse, 5′-ACAGGACTTGGGAGGTAT-3′; for CCND1 forward, 5′-GCGAGGAACAGAAGTGCG-3′ and reverse, 5′-TGGAGTTGTCGGTGTAGATGC-3′; for MYC forward, 5′-TCCTGTCCGTCCAAGCAG-3′ and reverse, 5′-ACGCACAAGAGTTCCGTAG −3′. The real-time PCR reactions were performed under the following conditions: denaturation at 95°C for 10 sec, annealing at 55°C for 30 sec, and extension at 72°C for 30 sec, a total of 35 cycles. The real-time PCR reactions were performed in the Roche 480 Real-Time PCR system with SYBR Premix Ex Taq™ according to the manufacturer's instructions.

Wound healing assay

Confluent GLAG-66 cell monolayers on 6-well tissue culture plastic dishes were transfected with pEnter-WNT10A, pEnter-LEF1 and their control plasmid pEnter-MOCK, as well as WNT10A and LEF1 siRNA. GLAG-66 cells were grown to confluent monolayers on 6-well plates and a thin disposable tip was used to create linear scratch wounds. Cultures were rinsed with PBS and replaced with fresh quiescent medium containing 10% fetal bovine serum. Wound images were taken with a digital camera mounted on a light microscope at 0, 24, 36 and 72 h. The wound gap widths were measured using ImageJ software.

Protein extraction and western blot analysis

Total cellular or tissue protein extracts were obtained using PMSF with RIPA (the ratio of PMSF to RIPA is 1:100). Cells or tissues were washed twice with cold PBS and lysed in a buffer containing 100 µl RIPA and 1 µl PMSF. Protein concentration was determined by the BCA method using the BCA protein assay (Thermo Scientific/Pierce, Courtaboeuf, France). Cellular or tissue protein extracts were separated on 10% SDS-PAGE gel. Proteins were transferred to polyvinylidene difluoride microporous membranes (Millipore, MA, USA), and then blocked with 5% non-fat dry milk for 1 h at room temperature. The membranes were incubated overnight at 4°C with the primary antibody specific to WNT10A (rabbit polyclonal, Abcam, 1:500, Abcam), β-catenin (rabbit monoclonal, 1:5,000, Abcam), LEF1 (rabbit monoclonal, 1:1,000, Abcam), MYC (rabbit monoclonal, 1:10,000, Abcam), cyclin D1 (rabbit monoclonal, 1:10,000, Abcam) or GAPDH (rabbit polyclonal, 1:500 Boster Co., Ltd., Wuhan, China) as the internal control. The membranes were washed and incubated with anti-rabbit antibody (rabbit polyclonal, 1:2,000 Boster) for 1 h at room temperature, which were subsequently washed and shown using chemiluminescence reagent.

Cell proliferation assay

A total of 2×103 cells were cultured in 96-well plates. Cell proliferation was quantified using the Cell Counting Kit-8 assay (Roche, Penzberg, Germany) according to the manufacturer's instructions. Absorbance was measured on a microplate reader (Tecan Sunrise, Mannedorf, Switzerland) at a wavelength of 450 nm. Data represent the average values of three independent experiments.

Apoptosis analysis

5×105 GLAG-66 cells in 6-well plates were transfected with pEnter-WNT10A, pEnter-LEF1, control plasmid pEnter-mock, WNT10AsiRNA and LEF1siRNA, and incubating for varied time-points before the cells were digested and harvested by centrifugation. Then the cells were separately fixed gently (drop by drop) in 70% ethanol overnight at −20°C and then re-suspended in 535 µl DDW containing 500 µl PI stain buffer, 10 µl RNaseA solution (50X) and 25 µl PI. After 30 min at 37°C in the dark, the cells were analyzed with flow cytometry equipped with an argon laser at 488 nm. Then cell cycle was determined and analyzed. Analysis of apoptosis was quantified by Annexin V-FITC apoptosis detection kit according to the manufacturer's instructions (BD Biosciences, USA) (21,22). Apoptotic events were acquired and analyzed by BD-FACS calibur instrument (BD Biosciences).

Results

WNT10A/β-catenin signaling pathway is activated in papillary thyroid cancer

To investigate the molecular mechanisms involved in tumorigenesis of papillary thyroid cancer, microarray analysis was performed to compare the variation of gene expressions within the PCT cell populations and their adjacent normal tissues. Up- or downregulation 1.5-fold was set as a cutoff value, and changes in gene expression were analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID). In the DAVID analysis, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway related to papillary thyroid cancer was hsa05216: thyroid cancer, six genes (CCND1, RXRG, LEF1, MYC, CTNNB1 and TPM3) were upregulated >1.5-fold in this pathway (Fig. 1A). All these genes were associated with WNT/β-catenin signaling pathway, among them LEF1 and CTNNB1 play critical roles in WNT/β-catenin signaling pathway. Of the signaling proteins, WNT10A was the only WNT ligand that was upregulated >1.5-fold and its upregulation was found to be >4-fold in the papillary thyroid cancer comparing to adjacent normal tissues.

Figure 1. WNT10A/β-catenin signaling pathway is activated in microarray analysis data of papillary thyroid cancer and adjacent normal tissues. Microarray analysis showed that CCND1, RXRG, LEF1, MYC, CTNNB1, TPM3 expression increased >1.5-fold in papillary thyroid cancer tissues, all these molecules are involved in WNT10A/β-catenin signaling pathway. (A) Upregulated expression of the molecules involved in WNT10A/β-catenin signaling pathway was verified by QRT-PCR (B). QRT-PCR was performed as described in Materials and methods.

Molecules involved in WNT10A/β-catenin signaling pathway are upregulated in papillary thyroid cancer tissues

Next, the expression of WNT10A, LEF1, MYC, β-catenin and cyclin D1 were verified by QRT-PCR in 15 cases of papillary thyroid cancer (Fig. 1B). The real-time PCR (QRT-PCR) products were further separated by agarose gel electrophoresis and visualized to compare the initial levels of WNT10A, LEF1, MYC, β-catenin and cyclin D1 mRNA in the tissues from papillary thyroid cancer and adjacent normal tissues (Fig. 2A). To confirm the results at mRNA level, WNT10A, LEF1, MYC, β-catenin and cyclin D1 expression was further detected by western blot analysis in 10 cases of papillary thyroid cancer and adjacent normal tissues, the results demonstrated upregulated expression in papillary thyroid cancer tissues comparing to adjacent normal ones (Fig. 2B). The results demonstrated that WNT10A/β-catenin signaling pathway was activated in papillary thyroid cancer tissues.

Figure 2. Upregulated expression of the molecules involved in WNT10A/β-catenin signaling pathway verified by electrophoresis (A) and western blot analysis (B). (B) Lane 1, papillary thyroid cancer tissues; lane 2, adjacent normal tissues; lane 3, papillary thyroid cancer tissues; lane 4, adjacent normal tissues. QRT-PCR and western blot analysis were performed as described in Materials and methods.

WNT10A/β-catenin signaling pathway activation promotes proliferation of thyroid cells

To investigate the role of WNT10A/β-catenin signaling pathway in mediating migration and invasion of thyroid cancer cells, cultured GLAG-66 cells were transfected with pEnter-WNT10A, pEnter-LEF1 and control plasmid pEnter-mock. WNT10A siRNA and LEF1siRNA were employed to knockdown the expression of the two genes, and the proliferations of the treated cells were quantified with the Cell Counting Kit-8 assay. The expression of WNT10A in the cell lines was confirmed by RT-PCR and western blot analysis (Fig. 3A), the levels of β-catenin, LEF1, MYC and cyclin D1 protein were induced (Fig. 3B), suggesting that WNT10A effectively activates β-catenin signaling pathway in thyroid cancer cells. As a response to the WNT/β-catenin signaling pathway activation, the CCK-8 assay results showed that the WNT10A and LEF1 overexpressing cells exhibited significant higher level of proliferation as compared with the empty vector controls and WNT10A siRNA (Fig. 3C).

Figure 3. WNT10A/β-catenin signaling pathway activation promotes proliferation of thyroid cells. (A) QRT-PCR and western blot analysis of WNT10A expression under the overexpression plasmid pEnter-WNT10A and siRNA transfection in GLAG-66 cells. (B) Western blot analysis of β-catenin, MYC, cyclin D1 and LEF1 expression. Lane 1, 4 µg of pEnter-LEF1; lane 2, 4 µg of pEnter-LEF1 and WNT10A siRNA; lane 3, 4 µg of pEnter-WNT10A; lane 4, 4 µg of pEnter-WNT10A and LEF1siRNA; lane 5, control plasmid pEnter-mock. (C) Cell Counting Kit-8 assay of GLAG-66 cells (***P<0.0001, **P=0.0079; Student's t-test). QRT-PCR, western blot analysis and CCK-8 were performed as described in Materials and methods.

WNT10A/β-catenin signaling pathway activation suppressed late apoptosis of thyroid cancer cells

GLAG-66 cells were then examined by flow cytometry to test whether cell cycle and cell apoptosis were modulated concomitant with WNT10A and LEF1 administration. Following transfection with the pEnter-WNT10A and pEnter-LEF1 plasmid, respectively. The results indicated a lower percentage of late apoptotic cells after WNT10A and LEF1 overexpression (Fig. 4A, Q2-4). As expected, higher percentage of late apoptotic cells were detected after reduction of WNT10A and LEF1 with siRNA (Fig. 4B, Q2-4). Quantification of the results confirmed the experimental conclusion (Fig. 4C).

Figure 4. WNT10A/β-catenin signaling pathway activation suppresses late apoptosis of thyroid cancer cells. (A) Late apoptotic cells were reduced concomitant with pEnter-WNT10A and pEnter-LEF1 transfection. (B) Late apoptotic cells were restored by pEnter-WNT10A and pEnter-LEF1 siRNA. (C) Quantification of apoptosis analysis results (Student's t-test). RNA interference and apoptosis analysis were performed as described in Materials and methods.

WNT10A/β-catenin signaling pathway activation enhances migration of the thyroid cells

Next, the directional migration of GLAG-66 cells was examined with the wound-healing assay under WNT10A/β-catenin signaling pathway activation. The ‘wound’ was created in confluent cell cultures (0 h), and migration of cells into the gap was monitored after 72 h. As shown in Fig. 5A, GLAG-66 cells grew into the wound as time passed, and the speeds of the WNT10A and LEF1 overexpressing cells migrating into the wound were significantly increased compared to the control cells. In line with this result, knockdown of WNT10A and LEF1 expression with their siRNA reduced the migration ability of GLAG-66 cells, respectively. Quantification of wound healing assay confirmed this conclusion (Fig. 5B). These data showed that upregulation of WNT10A and LEF1 expression significantly increased migration of the GLAG-66 cells.

Figure 5. WNT10A/β-catenin signaling pathway activation enhanced migration of the thyroid cells. (A) WNT10A and LEF1 expression in GLAG-66 cells had significant effects on cell migration; cell migration was enhanced when WNT10A and LEF1 were overexpressed, whereas migration was reduced as WNT10A and LEF1 were downregulated. (B) Quantification of wound healing assay results (Student's t-test). Wound healing assays were performed as described in Materials and methods.

Discussion

WNT/β-catenin signaling is known to be an important factor in stem cell regulation of intestinal cancer (23,24), breast cancer (25–27) and skin cancer (28). Activation of canonical WNT signaling pathway is an important process both in the establishment and maintenance of cancer stem cells (29), as well as promoting self-renewal phenotype (18). Previous studies have indicated that DACT2 (30), PPFP (31), Dickkopf-1 (32), GPX3 (33), miR-146b-5p (34) and PROX1 (35) are methylated or disordered in thyroid cancer, leading to growth, metastasis and EMT of thyroid cancer cells by activating WNT signaling pathway. The WNT/β-catenin pathway is a direct and forward enhancer of the TTF-1 expression that can regulate serum thyroglobulin levels, which is intimately linked to follow-up of PTC patients (5,36).

In previous studies, WNT10A was shown to play an important role in the pathogenesis of idiopathic pulmonary fibrosis (37), agenesis of the maxillary permanent canines and dental agenesis (38,39), hypohidrotic ectodermal dysplasia (40) and keratoconus (41). Specifically, the aberration of WNT10A leads to malformation of ectodermal appendages during development. During hair follicle morphogenesis, expression of WNT10A is weakly upregulated in the placode compared with adjacent epidermis (42). Actually, WNT10A uniquely observed in basal and mammary stem cells may regulate the canonical WNT signaling pathway to maintain basal and mammary stem cell activity and was secreted by mammary stem cells (43). Consequently, it is believed that WNT10A may be necessary for epithelial migration and proliferation during normal development. However, the role of WNT10A in PTC is still unknown.

By employing microarray analysis of PTC, we found that WNT10A was upregulated in PTC tissues but limitedly expressed in adjacent normal tissues. WNT10A and WNT6 may be considered to be the key factors in human carcinogenesis through activation of WNT/β-catenin pathway (44). Previous studies have shown that WNT6 expression is not significantly different in primary gastric cancer. However, another report claimed that WNT10A was upregulated in primary gastric cancer (45). It was further verified that overexpression or knockdown of WNT10A had direct influence on proliferation, migration and invasion of PTC cells by modulating WNT/β-catenin pathway in thyroid papillary cancer. In our study, it was found that overexpression of WNT10A led to increase in β-catenin, upregulated cyclin D1 and c-myc expression and it was the same with overexpression of LEF1. Forced WNT10A expression in GLAG-66 cells resulted in increased cell proliferation, migration, invasiveness, and cell transformation. Consistently, knockdown of WNT10A in GLAG-66 can decrease intracellular β-catenin accumulation, downregulate cyclin D1 and c-myc expression, and thus suppress cell proliferation, migration, invasiveness, and cell transformation. Combined with the designs of cell line models, our results suggested that WNT10A might exert an autocrine effect on PTC, resulting in activation of WNT/β-catenin signaling and promoting cell proliferation and migration.

These data indicated that WNT10A plays a crucial role in carcinogenesis and aggressiveness in papillary thyroid cancer by activating β-catenin-dependent pathway. This in turn would also increase β-catenin, LEF1, MYC and cyclin D1 expression. Similar results were obtained from esophageal squamous cell carcinoma, the upregulation of WNT10A has been proven to induce proliferation and migration and to promote a self-renewal phenotype of esophageal carcinoma cell lines (18). Several molecular components in Wnt/β-catenin signaling have been proposed as potential therapeutic targets of cancer treatment (46). Wnt/β-catenin signaling is associated with lymph node metastasis of PTC by regulating the expression of cyclin D1 (47). Dickkopf-1, a Wnt/β-catenin pathway inhibitor, suppressed proliferation and migration of PTC cells by modulating the Wnt/β-catenin pathway (48). Our study demonstrated the role of WNT10A/β-catenin pathway in thyroid tumorigenesis for the first time. WNT10A/β-catenin shows potential as a future therapeutic target for papillary thyroid cancer.

Acknowledgments

We gratefully acknowledge the financial support from The National Natural Science Foundation of China (81272351), The Fundamental Research Funds of Shandong University (2015JC010), The Project of Medical and Health Technology Development Program in Shandong Province (grant no. 2014ws0349), The Open Research Fund of State Key Laboratory of Environmental Chemistry and Ecotoxicology (KF2014-08) and The Primary Research and Development Plan of Shandong Province (2016GSF201137).

References