β-Catenin Attenuation Inhibits Tumor Growth and Promotes Differentiation in a BRAF^V600E-Driven Thyroid Cancer Animal Model

Papillary thyroid cancer (PTC) is the most common type of thyroid cancer, accounting for more than 80% of thyroid cancer cases (1). Although PTC has excellent prognosis with less than 3% mortality at 10 years after treatment, more than 25% of patients developed recurrence during long-term follow-up, which remains a major problem for patients with thyroid cancer (2, 3). In some studies, BRAF^V600E mutation has been shown to be one of the factors contributing to thyroid cancer recurrence and mortality (4, 5).

BRAF^V600E mutation is the most frequent genetic alteration in PTC occurring in about 45% of cases (6, 7). The mutation constitutively activates the RAS–RAF–MEK–ERK MAPK pathway and promotes initiation and growth of PTC. It has been demonstrated that Braf^V600E drives oncogenic transformation of thyroid epithelial cells and development of PTC in transgenic mouse models (8, 9).

The Wnt/β-catenin pathway plays an important role in embryonic development and tissue homeostasis (10). Dysregulation of its signaling is associated with many types of disease including cancer (11). β-Catenin is a key downstream component of both cadherin–catenin cell adhesion complex and Wnt signaling pathway (12, 13). The dual functional protein is involved in the regulation of cadherin-mediated cell–cell adhesion and transcriptional coactivation of Wnt target genes such as MYC (c-Myc), JUN (C-Jun), FOSL1 (Fra-1), CCND1 (cyclin D1), and ABCB1 (multidrug resistance protein 1) via TCF/LEF family of DNA-binding proteins (14, 15).

Aberrant β-catenin expression or localization has been reported in patients with PTC and is associated with c-Myc and cyclin D1 overexpression (16, 17). Frequent activating CTNNB1 mutations and β-catenin nuclear localization have also been reported in the early studies of poorly differentiated or anaplastic thyroid carcinoma (ATC; refs. 18, 19). However, recent studies using the next-generation sequencing are unable to demonstrate frequent CTNNB1 mutations in ATC (20). The functional interactions between β-catenin and MAPK, PI3K/AKT, or CREB (cAMP-response element binding protein) signaling pathways have been shown to promote cell proliferation in thyroid follicular cells and cancer cell lines (21, 22). These early studies suggest that RAS- or BRAF-driven PTC may require Wnt/β-catenin pathway to sustain its growth (23). However, it is not clear to what extent the aberrant β-catenin expression contributes to thyroid cancer development and growth in vivo, especially in the presence of BRAF^V600E mutation.

In this study, we used a mouse model of Braf^V600E-induced PTC to investigate the role of β-catenin in thyroid cancer growth. We found β-catenin knockout lead to significant reduction in tumor growth, increased survival, and sensitivity to BRAF^V600E inhibitor PLX4720.

PKF118–310 (β-catenin/KDM4A inhibitor, Catalog No. K4394) was obtained from Sigma-Aldrich (24), and PLX4720 (BRAF^V600E inhibitor, Catalog No. S1152) was from Selleck Chemicals (25). Antibodies were purchased from Cell Signaling Technology, Inc.: β-catenin (No. 8480), phospho-Erk ½ (No. 4370), phosphor-Akt (No. 4060), E-cadherin (No. 3195), Slug (No. 9585), Snail (No. 3879), and Vimentin (No. 5741).

The conditional Braf^V600E knock-in and β-catenin knockout mice (Ctnnb1^null) were created as described previously (26, 27). Thyroid-specific activation of mutant Braf^V600E allele was achieved by crossing with TPO-Cre deleter mice (28, 29). TPO-Braf^V600E mice with WT Ctnnb1 (BVE-Ctnnb1^WT or BVE) developed PTC at about 5 weeks of age and were used as PTC tumor controls. TPO-Braf^WT mice with WT Ctnnb1 were used as normal controls. TPO-Braf^V600E mice with thyroid-specific Ctnnb1 knockout (BVE-Ctnnb1^null) were obtained by several rounds of breeding among LSL-Braf^V600E (26), TPO-Cre (29), and floxed Ctnnb1 mice (27). To knockout Ctnnb1 in the thyroid of BVE mice, floxed Ctnnb1 mice were first crossed with LSL–Braf^V600E or TPO–Cre mice to generate floxed Ctnnb1^+/−::Braf^V600E strain or floxed Ctnnb1^+/−::TPO–Cre strain. The floxed Ctnnb1^+/−::Braf^V600E and floxed Ctnnb1^+/−::TPO–Cre mice were then bred together to generate TPO–Braf^V600E::Ctnnb1^null (BVE-Ctnnb1^null) mice, which had thyroid-specific activation of Braf^V600E and deletion of β-catenin via Cre-mediated recombination in the thyroid. Mice were housed in autoclaved filter-top cages with autoclaved food and water ad libitum, and maintained on a 12-hour light/12-hour dark cycle (6:00 AM to 6:00 PM). Mice of both sexes were used in the study, and similar results were obtained. The study was approved by the Animal Care and Use Committee of King Faisal Specialist Hospital and Research Centre (RACNo. 2190004) and conducted in compliance with the Guide for the Care and Use of Laboratory Animals (National Research Council).

Genotyping of Cre-mediated recombination of LSL–Braf^V600E mutant allele has been described previously (9, 28). Briefly, primers A (5′-AGTCAATCATCCACAGAGACCT-3′) + C (5′-GCCCAGGCTCTTTATGAGAA-3′) detect both WT Braf (466 bp) and Cre-recombined mutant Braf^V600E (518 bp) alleles, respectively. Primers B (5′-GCTTGGCTGGACGTAAACTC-3′) + C detect LSL–Braf^V600E allele (140 bp). Primers D (5′-GTTCGCAAGAACCTGATGGACA-3′) and E (5′-CTAGAGCCTGTTTTGCACGTTC-3′) detect Cre gene (350 bp). For genotyping of Cre-mediated recombination of floxed Ctnnb1 allele (27), primers 41 (5′-AAGGTAGAGTGATGAAAGTTGTT-3′) + 42 (5′-CACCATGTCCTCTGTCTATTC-3′) detect both WT (221 bp) and floxed (324 bp) Ctnnb1 alleles, respectively; primers 68 (5′-AATCACAGGGACTTCCATACCAG-3′) + 69 (5′-GCCCAGCCTTAGCCCAACT-3′) detect Cre-recombined deleted Ctnnb1 allele (631 bp). Genomic DNA was isolated from mouse tails or tumor tissues. The PCR conditions were as follows: 94°C for 5 minutes followed by 35 cycles of amplification (94°C for 30 seconds, 58°C for 30 seconds, 72°C for 1 minute) with a final extension at 72°C for 10 minutes.

Histology and IHC staining was performed as described previously (9). Briefly, 4-μm-thick formalin-fixed paraffin-embedded tissue sections were prepared and stained with hematoxylin and eosin (H&E) or with β-catenin antibody (1:100 dilution; Rabbit mAb No. 8480, Cell Signaling Technology, Inc). A DAKO LSAB + kit with horseradish peroxidase (HRP) was used for immunostaining (DAKO). The sections were counterstained with Mayer's hematoxylin.

Blood was collected by cardiac puncture. Serum TSH and T4 were measured using MILLIPLEX MAP Mouse Pituitary Magnetic Bead Panel (MPTMAG-49K) and Multi-Species Hormone Magnetic Bead Panel (MSHMAG-21K), respectively, following the manufacturer's instructions (EMD Millipore Corporation).

Thyroid tumors were collected aseptically from donor mice (BVE and BVE-Ctnnb1^null) using blunt dissection, mechanically dissociated by mincing and passing through a 40-μM mesh sterile screen, and suspended in DMEM/F12 growth medium (10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin) as described previously (28). Cells were cultured in DMEM/F12 growth medium containing 4 mU/mL bovine TSH (Sigma-Aldrich). For establishment of immortalized cell lines, cells were cultured continually for more than 6 months and passaged weekly or biweekly at 1:3 ratio with 0.25% trypsin. BVE-Ctnnb1^WT (BVE) and BVE-Ctnnb1^null cell lines had about 25 and 20 passages, respectively before they were used for experiments. Thyroid origin of the cell lines was confirmed by genotyping to rule out fibroblast contamination. Genotyping of the cell lines and expression of genes for thyroid hormone synthesis were verified every 6 months when the cells were in culture.

Total RNA from WT control thyroid tissues and thyroid tumors (BVE and BVE-Ctnnb1^null) were isolated by TRI Reagent solution (No. T9424, Sigma-Aldrich). Libraries were constructed using an Illumina TruSeq RNA Library Prep Kit according to the manufacturer's procedure. All sequencing was performed on Illumina Hiseq 4000 with at least 20 million clean reads. The significant DEGs were selected on the basis of the following criteria: Log2-fold change > 2, FDR < 0.001, and P value from difference test <0.01.

Mice at 4 months of age were anesthetized with 5% isoflurane mixed with oxygen, and maintained at 1.5% isoflurane during the scan. Mice were then given 5 MBq (megabecquerel) ¹²⁴I-labeled NaI solution by tail vein injection. Thyroid ¹²⁴I uptake from WT control, BVE, and BVE-Ctnnb1^null mice was measured at different time intervals (30 minutes, 2 hours, and 24 hours) by nanoScan PET/CT (Mediso Medical Imaging Systems).

Cell lysates were obtained by extraction in RIPA buffer (20 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 5 mmol/L EDTA, 1% NP-40) containing Pierce's Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific). Protein concentration was determined by Bradford's assay using a Bio-Rad Protein Assay Kit (Bio-Rad). Proteins (20–40 μg) were loaded onto a 12% SDS-PAGE and were transferred to a PVDF membrane. Western blot analysis was performed using antibodies (1:1,000 dilution, Cell Signaling Technology, Inc.) against β-catenin (No. 8480), phospho-Erk ½ (No. 4370), phosphor-Akt (No. 4060), E-Cadherin (No. 3195), Slug (No. 9585), Snail (No. 3879), and Vimentin (No. 5741).

BVE-Ctnnb1^WT and BVE-Ctnnb1^null cells were seeded in 6-well plates (10⁵ cells/well), respectively, and a linear scratch was created with a sterile pipette tip when the cells reached confluent monolayer. The cells were rinsed three times with medium to remove cellular debris. Cell migration or wound-healing were monitored by microscopy after 16-hour culture.

BVE-Ctnnb1^WT cells were cultured with different concentrations of PKF118–310, PLX4720, or both for 24 hours. The apoptosis was analyzed by flow cytometer using Vybrant Apoptosis Assay Kit (Molecular Probes).

BVE-Ctnnb1^WT and BVE-Ctnnb1^null cells were plated into 12-well plates (5 × 10² cells/well), respectively and cultured for 14 days in the presence of different concentrations of PLX4720, PKF118–310, or both. Cells were then fixed with methanol for 10 minutes and stained with 0.5% crystal violet dye in methanol:de-ionized water (1:5) for 10 minutes. After three washes with de-ionized water to remove excess crystal violet dye, the crystal violet dye was released from cells by incubation with 1% SDS for 2 hours before optical density (OD)^{570 nm} measurement.

BVE mice at 2 months of age were equally divided into four groups. Group 1 (n = 5) received an intraperitoneal (i.p.) injection of 2.5 μg PKF118–310 (0.4 mg/kg) dissolved in 50-μL phosphate-buffered saline pH7.4 (PBS), three times per week (day 1, 3, 5) for 2 months; group 2 (n = 5) received a daily i.p. injection of PLX4720 (20 mg/kg) for 2 months. PLX4720 was first dissolved in DMSO, followed by PBS (100 μL), which was then injected into mice; group 3 (n = 5) received an alternate i.p. injection of PKF118–310 (0.4 mg/kg, day 1, 3, 5) and PLX4720 (20 mg/kg, day 2, 4, 6) for 2 months; group 4 (n = 5) received an i.p. injection of vehicle (PBS) only. At the end of 2-month treatment, the mice were sacrificed and thyroids were harvested for histology analysis. Kaplan–Meier analysis of survival were performed in BVE-Ctnnb1^WT mice with or without treatment (n = 12 in each group).

Student t test (two-tailed) was used to compare two groups and one-way ANOVA was used to compare multiple groups. A P value of 0.05 or less was considered significant.

We first examined β-catenin (Ctnnb1) expression in normal thyroids from WT control mice (n = 10, pooled RNA) and Braf^V600E-induced PTC (n = 3) by RNA-seq analysis. Consistent with previous findings in human PTC, β-catenin expression was increased by more than threefold in both early and late stages of PTC (Fig. 1A, P < 0.001). This was confirmed by IHC of strong cytoplasmic staining (Fig. 1B-b). To investigate the impact of β-catenin overexpression on Braf^V600E-mediated thyroid tumorigenesis and growth, we knocked out Ctnnb1 gene by cross-breeding floxed Ctnnb1^−/−::Braf^V600E with floxed Ctnnb1^−/−::TPO–Cre mice. The Ctnnb1 knockout was confirmed by genotyping (Fig. 1C) and absence of β-catenin staining by IHC (Fig. 1D-b). As shown in Fig. 2A-c and d, BVE-Ctnnb1^null tumor growth was slowed and tumor was mainly located in the center of thyroid lobe with reduced papillary architecture, whereas BVE-Ctnnb1^WT tumor could be seen occupying almost the entire thyroid lobe with dominant papillary architecture at 4 months of age (Fig. 2A-a and b). The tumor weight was significantly reduced: 0.035 ± 0.004 g in BVE-Ctnnb1^null versus 0.063 ± 0.008 g in BVE mice (P < 0.05; Fig. 2C). Similar results were observed in the late stage of BVE-Ctnnb1^null tumors (10 months of age): 0.106 ± 0.02 g in the BVE-Ctnnb1^null versus 0.207 ± 0.04 g in the BVE mice (P < 0.05; Fig. 2C). The reduction in tumor growth was more apparent morphologically in the late stage of BVE-Ctnnb1^null tumors: reduction of papillary tumor volume, more follicular architecture, and many lymphocytes and macrophage infiltration for possible tumor clearance (Fig. 2B-c and d) whereas in the BVE mice, the entire thyroid bed was occupied by papillary tumor with few lymphocytes and macrophage infiltration (Fig. 2B-a and b). The bodyweight of BVE-Ctnnb1^null mice was increased by 20% (12.82 ± 0.98g, n = 16 at 4 months of age) as compared with BVE mice (10.28 ± 0.66 g, n = 14, P < 0.05), but was still 50% less than WT control mice (24.58 ± 0.76 g, n = 16). Although thyroid function (serum TSH and T4) was improved in the BVE-Ctnnb1^null mice, they still had severe hypothyroidism with high level of serum TSH and low level of T4 (Fig. 2D). Taken together, these data indicate Ctnnb1 deletion reduces tumor growth and promotes differentiation, but it is not sufficient to reverse hypothyroidism caused by Braf^V600E mutation.

To further assess the effect of Ctnnb1 knockout on the differentiation of thyroid follicular cells, we analyzed the expression of genes involved in the thyroid hormone synthesis from WT control thyroids, BVE and BVE-Ctnnb1^null tumors by RNA-seq analysis. As shown in Fig. 3A, the expression of genes involved in the thyroid hormone synthesis were significantly downregulated in the BVE tumors (n = 3) as compared with WT control thyroids (n = 10): 80-fold decrease in reads per kilobase per million reads (RPKM) in Tg (529.22 ± 141.41 vs. 42398.8 ± 1499.36), 14-fold decrease in Tpo (47.56 ± 15.83 vs. 691.94 ± 41.08), twofold decrease in Tshr (4.25 ± 1.33 vs. 10.73 ± 6.3), 70-fold decrease in Duox2 (0.22 ± 0.04 vs. 14.08 ± 2.75), 22-fold decrease in Duoxa2 (1.71 ± 0.4 vs. 38.7 ± 9.07), 56-fold decrease in Slc5a5 (4.8 ± 1.39 vs. 269.79 ± 39.51), 72-fold decrease in Slc5a8 (0.51 ± 0.07 vs. 36.25 ± 0.25), 47-fold decrease in Slc26a7 (2.29 ± 0.37 vs. 108.76 ± 63.31), and sixfold decrease in Iyd (42.89 ± 3.74 vs. 241.3 ± 103.12). The deletion of Ctnnb1 gene resulted in upregulation of these genes in the BVE-Ctnnb1^null tumors as compared with the BVE tumors. Slc5a5, the sodium-iodide symporter (NIS) for basolateral iodide transport into thyroid follicular cells, was the most upregulated gene with 18-fold increase in its expression (Fig. 3B; Supplementary Fig. S1). However, Ctnnb1 deletion only partially rescued these downregulated genes up to 40% (Fig. 3B). There are four genes involved in the apical iodide efflux into the follicular lumen: Slc26a7 (30–32), Slc26a4 (33), Slc5a8 (34), and Ano1(35). In contrast to Slc26a7 whose expression was downregulated by 47-fold in the BVE tumors, the expression of Slc26a4 and Ano1 was increased by seven- and fourfold, respectively (Fig. 3C), which may compensate the loss of Slc26a7 function and mitigate the deficiency in apical iodide efflux. Finally, we evaluated thyroid ¹²⁴I uptake by PET-CT scan to see if elevated Slc5a5 expression correlated with increased iodine uptake. As shown in Fig. 3D, ¹²⁴I uptake in the BVE-Ctnnb1^null mice was comparable with the WT mice and modestly higher than the BVE mice at 30 minutes and 2 hours intervals following tail vein injection of 5 MBq ¹²⁴I-labeled NaI solution. The high concentration of ¹²⁴I and large tumor size may contribute to the insignificant difference in the early ¹²⁴I uptake. At 24 hours interval, ¹²⁴I uptake was about threefold higher in the BVE-Ctnnb1^null mice than that in the BVE mice (0.065 ± 0.005 vs. 0.025 ± 0.005 MBq, n = 3), but was still threefold lower than the WT mice (0.223 ± 0.017, n = 3; Fig. 3D). Thus, ¹²⁴I measurement at 24 hours is more sensitive to detect deficiency in the iodine uptake. These dada confirm that increased Slc5a5 expression results in elevated iodide uptake in the BVE-Ctnnb1^null mice.

Increased expression of p-Erk, p-Akt, Snail, Slug, and vimentin is associated with thyroid cancer growth and progression (36, 37). Both Snail and Slug are zinc finger transcription factors and downstream targets of TGFβ, which promotes epithelial–mesenchymal transition (EMT) by downregulating the expression of adhesion molecule E-cadherin (38). Therefore, to investigate the mechanisms leading to thyroid tumor growth inhibition in the BVE-Ctnnb1^null mice, we studied the expression of p-Erk, p-Akt, Snail (Snail1), Slug (Snail2), vimentin, and E-cadherin in three BVE-Ctnnb1^null tumors and two cell lines established from BVE and BVE-Ctnnb1^null thyroid tumors by Western blot analysis. As shown in Fig. 4A, the expression levels of p-Erk and p-Akt were decreased in both BVE-Ctnnb1^null tumor samples and cell line, suggesting downregulation of MAPK and PI3K/Akt signaling pathways. As shown in Fig. 4B, both Snail and Slug expression levels were reduced as well. As expected, E-cadherin expression was increased and the expression of mesenchymal cell marker vimentin was decreased in both BVE-Ctnnb1^null tumor samples and cell line (Fig. 4B). Furthermore, cell migration was also reduced in the BVE-Ctnnb1^null cell line (Fig. 4C). The long-term survival of BVE-Ctnnb1^null mice was compared with BVE mice (n = 20 in each group). As shown in Fig. 4D, the survival of BVE-Ctnnb1^null mice was significantly increased and more than 50% of mice were still alive after 14-month observation (P < 0.0001). These data demonstrate that active β-catenin signaling is required in BRAF^V600E-mediated tumor growth.

To evaluate the sensitivity of tumor cells to PKF118–310 and PLX4720, BVE-Ctnnb1^WT and BVE-Ctnnb1^null cell lines were used for colony formation assay to measure the long-term effect of PKF118–310 and PLX4720 on cell proliferation. The cells were cultured in different concentrations of PKF118–310 or PLX4720 alone or in combination for 14 days. As shown in Fig. 5A, PKF118–310 alone at 0.5 μmol/L resulted in 24% decrease in cell viability of BVE-Ctnnb1^WT cells. However, it significantly enhanced antiproliferative effect of PLX4720, resulting in 73% decrease in cell viability (reduction of cell viability to 27%) after combined treatment of 1 μmol/L PLX4720 and 0.5 μmol/L PKF118–310 versus 16% decrease in cell viability (84% cells still viable) for 1 μmol/L PLX4720 alone or 24% decrease in cell viability (76% cells remain viable) for 0.5 μmol/L PKF118–310 alone (P < 0.0001, Fig. 5A). In the BVE-Ctnnb1^null cells, 0.5 μmol/L PKF118–310 treatment resulted in 40% cell death whereas 0.25 μmol/L PLX4720 lead to more than 80% cell death (Fig. 5A, P < 0.0001), indicating that BVE-Ctnnb1^null cells became more sensitive to PKF118–310 and PLX4720 inhibitors. Next, we investigated the combined treatment on apoptosis of BVE-Ctnnb1^WT cells. As shown in Fig. 5B, apoptosis was found in more than 70% of cells after combined treatment with 2 μmol/L PLX4720 and 1 μmol/L PKF118–310 for 24 hours.

Finally, we investigated PKF118–310, PLX4720, or combined treatment on thyroid tumor growth and differentiation in the BVE mice. As shown in Fig. 6A-b and c, thyroid tumor growth was slowed following PKF118–310 treatment and the effect was similar to PLX4720 treatment: reduction in tumor volume with many lymphocyte and macrophage infiltration. A further reduction in tumor volume and increased differentiation into follicular architecture were demonstrated after combined treatment of PKF118–310 and PLX4720 (Fig. 6A-d), which was comparable to BVE-Ctnnb1^null mice treated with PLX4720 (Fig. 6A-f). As shown in Fig. 6B, significant reduction in tumor load was observed in the PLX4720 (0.049 ± 0.004 g, P < 0.05) and PLX4720 + PKF118–310 (0.032 ± 0.005 g, P < 0.01) groups as compared with the BVE control (0.068 ± 0.007 g). Although there was reduction in tumor volume in the PKF118–310 group (0.054 ± 0.008 g), it was not statistically significant, probably due to small sample size. As shown in Fig. 6C and D, the survival of BVE mice was significantly increased following treatment of PKF118–310 (P < 0.05), or PLX4720 + PKF118–310 (P < 0.01). However, the difference in survival between PKF118–310 and PKF118–310 + PLX4720 groups was modest and not statistically significant (P = 0.33). This is probably due to short follow-up duration and small number of animals in each group. Significant difference may be demonstrated if longer follow-up was observed and more animals were used.

In this study, we have shown that β-catenin cooperates with oncogenic Braf^V600E to drive thyroid cancer growth. The oncogenic potential of BRAF^V600E is significantly reduced after Ctnnb1 knockout, resulting in tumor growth inhibition, increased sensitivity to BRAF^V600E inhibitor, elevated iodide uptake, and improved thyroid function. The therapeutic effect of BRAF^V600E inhibitor PLX4720 is further enhanced when dual β-catenin/KDM4A inhibitor PKF118–310 is used together.

The poor response to radioiodine ablation therapy after thyroidectomy is a major cause for thyroid cancer recurrence and poor prognosis (39). The loss of radioiodine avidity is due to aberrant silencing of iodide-metabolizing genes such as SLC5A5, TSHR, TPO, and TG in thyroid cancer cells. Mutant BRAF^V600E inhibits these genes by constitutive activation of MAPK and TGFβ pathways (40–42). Suppression of MAPK pathway could partially restore expression of iodide-metabolizing genes in BRAF^V600E mutant thyroid cancer cells (43). In this study, we have shown that β-catenin is involved in tumor growth and downregulation of iodide-metabolizing genes in Braf^V600E-induced thyroid cancer. β-catenin ablation partially reverses aberrant silencing of iodide-metabolizing genes. The effect is probably indirect and mediated through the reduction of p-Erk activation since its activation is significantly reduced following β-catenin ablation. Our data suggest that constitutive BRAF^V600E activation depends on active β-catenin signaling. These novel findings may offer a new therapeutic approach of combined β-catenin and BRAF^V600E inhibitors in the treatment of iodine-refractory thyroid cancer.

Thyroid Iodide efflux to the follicular lumen via apical membrane for thyroid hormone synthesis is less well known and may require several genes such as SLC26A4 (Pendrin; ref. 44), ANO1 (Anoctamin 1; ref. 35), SLC5A8 (34), and SLC26A7 (30, 31). In our murine model, the expression of Slc5a8 and Slc26a7 is down-regulated as a result of Braf^V600E mutation. Interestingly, mRNA levels of Slc26a4 and Ano1 are increased, which may compensate for the functional loss of Slc5a8 and Slc26a7. However, SLC26A7 may be the most important gene in the iodide efflux since its loss-of-function mutations result in severe hypothyroidism which cannot be rescued by normal SLC5A8, ANO1, and SLC26A4 genes (30, 32). It is well-known that SLC5A5 (NIS) is involved in iodide transport into the thyroid follicular cells. Our data have shown that increased Slc5a5 expression leads to elevated iodide uptake in the BVE-Ctnnb1^null mice. Iodotyrosine deiodinase (IYD) catalyzes iodide recycling and promotes iodide retention in the thyroid follicular cells. Given that its expression is reduced in both BVE (about 5% of WT control) and BVE-Ctnnb1^null tumors (about 12% of WT control) (Fig. 3A and B), reduced iodide recycling may also contribute to decreased iodide uptake in the BVE and to a lesser extent BVE-Ctnnb1^null mice. This may explain significant reduction in the iodide uptake in both BVE and BVE-Ctnnb1^null tumors as compared with normal thyroid at 24 hours: ¹²⁴I being excreted instead of recycled back into the thyroid.

The Wnt/β-catenin signaling pathway is frequently dysregulated in cancer. Extensive crosstalk exits between Wnt/β-catenin and MAPK signaling in cancer (45). The outcome between their interactions depend on the specific cellular context. For example, in melanoma, Wnt/β-catenin signaling may act as a tumor suppressor to promote programmed cell death: BRAF^V600E downregulates Wnt/β-catenin signaling cascade. Activation of Wnt/β-catenin signaling synergizes BRAF^V600E inhibitor PLX4720 to reduce melanoma growth and increase apoptosis via Wnt-mediated reduction of AXIN1 (46). In contrast, Wnt/β-catenin signaling functions as an oncogene in colorectal cancer: its activation leads to increased MAPK signaling through Ras stabilization and drives malignant transformation (47). In the Braf^V600E murine thyroid cancer, β-catenin expression is upregulated and its knockout results in downregulation of multiple signaling pathways such as MAPK, PI3K/Akt, and TGFβ, indicating a positive crosstalk between Wnt/β-catenin and these signaling pathways. Furthermore, active Wnt/β-catenin signaling is required for Braf^V600E-mediated tumor growth. Sastre-Perona and colleagues have demonstrated activation of β-catenin signaling by Hras^G12V but not Braf^V600E in PCCl3 rat thyroid follicular cells (22). In our study, Braf^V600E is involved in the activation of β-catenin. The use of different model systems (in vitro rat thyroid follicular cells vs. in vivo Braf^V600E-driven thyroid cancer) might contribute to the variation. Interestingly, Damsky and colleagues have reported that β-catenin is a central mediator of Braf^V600E-driven melanoma metastasis and regulates both MAPK and PI3K/Akt signaling (48).

Resistance to BRAF^V600E inhibitors after prolonged treatment is a major challenge in targeted cancer therapy. Activation of Wnt/β-catenin pathway is one of the mechanisms of resistance. Combined inhibition of both Wnt/β-catenin and MAPK signaling pathways has shown synergistic antitumor effects in BRAF^V600E-mutant colorectal cancer cell lines (49). This study also demonstrates Wnt/β-catenin pathway plays an important role in conferring resistance to BRAF^V600E inhibitor in Braf^V600E-mutant thyroid cancer. The resistance could be reduced by the dual β-catenin/KDM4A inhibitor PKF118–310, which was initially identified as an inhibitor of Tcf/β-catenin signaling by a high-throughput screening of natural compounds (24, 50). PKF118–310 interrupts the interaction between β-catenin and Tcf/Lef transcription factors and has been shown to induce apoptosis, cell-cycle arrest, and suppress in vivo tumor growth in several xenograft models (51–53). PKF118–310 has recently been described as an inhibitor of histone lysine demethylase 4A (KDM4A) in epigenetic histone regulation (54). KDM4A is known to function as an oncogene when it is overexpressed (55, 56). A twofold increase in Kdm4a expression was found in both BVE and BVE-Ctnnb1^null tumors and cell lines. Ctnnb1 ablation did not affect its expression in the BVE-Ctnnb1^null tumors (Supplementary Fig. S2). It is thus possible that Kdm4a overexpression may be involved in the tumor growth of both BVE and BVE-Ctnnb1^null mice. The in vitro effects of PKF118–310 on BVE-Ctnnb1^null cells (inhibition of cell proliferation) support KDM4A involvement in thyroid cancer growth. The reduction of tumor volume in vivo in the BVE mice may be through its dual inhibition of Tcf/β-catenin signaling and KDM4A-mediate epigenetic modifications. Given its dual inhibition functions and few KDM4-selective inhibitors available, this tool drug may be a good candidate for further development into a cancer therapeutic drug.

In summary, we have demonstrated Wnt/β-catenin signaling pathway plays an important role in thyroid cancer growth and differentiation. Dual β-catenin/KDM4A inhibitor PKF118–310 significantly increases the antitumor activity of BRAF^V600E inhibitor PLX4720 leading to prolonged survival of Braf^V600E-mutant thyroid cancer. Targeting both MAPK and Wnt/β-catenin pathways may have significant therapeutic benefit for BRAF^V600E inhibitor-resistant and/or radioiodine-refractory thyroid cancer.

No disclosures were reported.

M. Zou: Conceptualization, data curation, formal analysis, validation, investigation, visualization, methodology, writing–original draft, writing–review and editing. H.A. BinEssa: Formal analysis, validation, investigation, visualization, methodology, writing–review and editing. Y.H. Al-Malki: Data curation, investigation, methodology, writing–review and editing. S. Al-Yahya: Data curation, investigation, methodology. M. Al-Alwan: Data curation, investigation, methodology, writing–review and editing. I. Al-Jammaz: Data curation, investigation, methodology. K.S.A. Khabar: Data curation, investigation, writing–review and editing. F. Almohanna: Data curation, investigation, project administration. A.M. Assiri: Resources, data curation, project administration. B.F. Meyer: Resources, supervision, investigation. A.S. Alzahrani: Conceptualization, resources, supervision, writing–review and editing. F.A. Al-Mohanna: Conceptualization, resources, investigation, writing–review and editing. Y. Shi: Conceptualization, supervision, funding acquisition, validation, investigation, methodology, writing–original draft, writing–review and editing.

We would like to thank Ms. Roua A. Al-Rijjal, Ms. Anwar F. Al-Enezi, and Mr. Wilfredo Antiquera for excellent technical support; Drs. Shioko Kimura, Catrin Pritchard, and Rolf Kemler for generous gifts of TPO-Cre, LSL-Braf^V600E, and floxed Ctnnb1 mice, respectively; and Mr. Cong Li and Kai Huang from BGI for bioinformatics service. This study was supported by KACST grant 13-MED1765-20 to Y. Shi.

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