Melatonin synergizes BRAF‐targeting agent dabrafenib for the treatment of anaplastic thyroid cancer by inhibiting AKT/hTERT signalling

Abstract As a selective inhibitor of BRAF kinase, dabrafenib has shown potent anti‐tumour activities in patients with BRAFV600E mutant anaplastic thyroid cancer. However, the resistance of thyroid cancer cells to dabrafenib limited its therapeutic effect. The effects of melatonin and dabrafenib as monotherapy or in combination on the proliferation, cell cycle arrest, apoptosis, migration and invasion of anaplastic thyroid cancer cells were examined. The molecular mechanism involved in drug combinations was also revealed. Melatonin enhanced dabrafenib‐mediated inhibition of cell proliferation, migration and invasion, and promoted dabrafenib‐induced apoptosis and cell cycle arrest in anaplastic thyroid cancer cells. Molecular mechanistic studies further uncovered that melatonin synergized with dabrafenib to inhibit AKT and EMT signalling pathways. Furthermore, melatonin and dabrafenib synergistically inhibited the expression of hTERT, and the inhibition of cell viability and the induction of cell cycle arrest mediated by the combination of these two drugs were reversed by hTERT overexpression. Taken together, our results demonstrated that melatonin synergized the anti‐tumour effect of dabrafenib in human anaplastic thyroid cancer cells by inhibiting multiple signalling pathways, and provided new insights in exploring the potential therapeutic targets for the treatment of anaplastic thyroid cancer.

cancer (PTC), medullary thyroid cancer (MTC) and anaplastic thyroid cancer (ATC). Among them, FTC, PTC and ATC are derived from the follicular thyrocytes, while MTC is the only parafollicular C-cell-derived tumour. Approximately 90% of thyroid cancers are PTC and FTC, which are usually curable with a 10-year survival rate of more than 85%. 4 In addition, about 3% of thyroid cancer cases are MTC, and the overall 5-year survival rate is over 80%. 5 ATC is rare, accounting for only 1% to 2% of all thyroid cancers, but leads to the majority of thyroid cancer-related deaths, making it one of the most aggressive human malignancies. 6,7 Patients diagnosed with ATCs have a median survival of 3-6 months due to its aggressive growth, extrathyroidal invasion, distant metastasis and resistance to conventional treatment. 8,9 To date, there is no standard or effective therapy to prolong the survival of ATC patients. 7,10,11 Therefore, there is an urgent need to identify new biological targets that can be converted into therapeutic approaches.
Recently, several driver mutations have been identified in ATC, resulting in a better understanding of the development of this heterogeneous disease. 12 BRAF mutation, specifically BRAFV600E mutation, occurs in approximately 25% of ATC. 13 This mutation leads to constitutive activation of the BRAF kinase, followed by phosphorylation of MEK and ERK to induce oncogenic activation of the mitogen-activated protein kinase (MAPK) pathway which transmits mitogenic signals from the cell membrane to the nucleus and plays a critical role in mediating cellular proliferation, differentiation, apoptosis and survival. 7,14,15 Dabrafenib, an effective ATP competitive inhibitor which selectively inhibits the activity of BRAFV600E kinase, displays impressive initial response rates. 16,17 Unfortunately, the perdurability of response is limited owing to the drug resistance. 17,18 Therefore, overcoming the acquired resistance to BRAFV600E mutation inhibitors and understanding the underlying mechanisms are critical for improving patient outcomes.
Melatonin (N-acetyl-5-methoxytryptamine) is an indoleamine compound, most of which is synthesized and secreted by the pineal gland. It is well known that melatonin is involved in the regulation of circadian rhythms and endocrine functions. 19 Actually, melatonin has been reported to participate in many other functions, including antioxidation, anti-angiogenesis, anti-inflammatory and activation of the immune system. [20][21][22][23] In recent years, increasing evidence has demonstrated that melatonin has anti-tumour effect in various cancers, such as melanoma, breast cancer, lung carcinoma and thyroid cancer. [24][25][26][27] Melatonin has been shown to be an effective combination therapy for cancers by improving the curative effect of conventional anticancer drugs, reducing their side effects and overcoming their resistance to chemotherapy. 28,29 However, the combination of melatonin and BRAF-targeting agent dabrafenib for the treatment of anaplastic thyroid cancer has not been reported.
In this study, we evaluated the role of melatonin in enhancing dabrafenib-mediated anti-tumour effects and uncovered the potential molecular mechanisms of this combination therapy in anaplastic thyroid cancer.

| Cell lines and culture conditions
Human anaplastic thyroid cancer cell lines SW1736, OCUT1, KHM-5M and CAL-62 were all obtained from the American Type Culture Collection (ATCC). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM), which contained 10% foetal bovine serum and antibiotics (100 U/mL penicillin and 100 g/mL streptomycin). were purchased from Cell Signaling Technology (USA). The anti-hTERT antibody (#ab32020) was purchased from Abcam (USA), and anti-MMP-9 antibody (#AF5228) was purchased from Affinity Biosciences (USA).

| Western blot
Cells were lysed on ice in protein extraction reagent, and protein concentration was determined by using BCA Protein Assay Kit (Thermo Fisher Scientific, USA). Equal amounts of proteins were loaded onto 10%-15% gradient SDS-PAGE gels and then transferred onto polyvinylidene fluoride membranes. Western blots were immunoblotted with the specific primary antibodies, followed by incubation with HRP-conjugated secondary antibody, finally detected by enhanced chemiluminescence.

| Cell viability assay
Thyroid cancer cells were seeded into 96-well plates (4000-6000 cells/well) and cultured with different treatment. Cell viability was assessed by the CCK-8 assay (Apexbio, USA), and the absorbance was measured at the wavelength of 450 nm.
Synergistic effect assessment of melatonin and dabrafenib was based on cell viability. The combination index (CI) values, using Chou and Talalay methods, have been widely used to characterize drug interactions. CI ˂1, =1 and ˃1 represented synergism, additive and antagonism effects, respectively.

| Colony formation assay
Thyroid cancer cells treated with the indicated doses of melatonin or dabrafenib were harvested and counted. Approximately 1000 cells were seeded in triplicate into 6-well plates and incubated for 8-14 days until grew into macroscopic colonies. Then, the cells were washed with PBS solution, fixed for 15 minutes and stained with 0.1% crystal violet for 20 minutes. The clones that contained more than 50 cells were counted and photographed.

| Scratch assay
Scratch assay was used to detect cell migration ability. SW1736 and OCUT1 cells were plated in 6-well plates and grown to a confluence of approximately 70%. The cells were then scraped in a straight line to create a 'scratch' and treated with melatonin or dabrafenib, alone or in combination. The wound gap was photographed by inverted microscope at 0 and 48 hours.

| Transwell invasion assay
1 × 10 4 thyroid cancer cells with indicated treatment were suspended in 100 μL serum-free medium and added to each matrigel-coated upper chamber (Corning, USA), while the lower chambers were filled with 500 μL medium containing 20% foetal bovine serum. After incubation at 37°C for 24 hours, the cells that did not invade through the pores were removed with a cotton swab. The chambers were washed, fixed, stained with 0.1% crystal violet and counted in three random view fields.

| Cell cycle assay
Thyroid cancer cells were collected after treatment with melatonin or dabrafenib, and cell cycle was analysed using a Cell Cycle Detection Kit (KeyGen Biotech, China). Cells were sorted by FACSCanto II Flow Cytometer (BD Biosciences, USA), and the relative proportions of cells in the G1, S and G2-M phases of the cell cycle were analysed by using FlowJo 7.6 software.

| RNA extraction and real-time qPCR
Total RNA was extracted from thyroid cancer cells by using TRIzol reagent (Life Technologies, USA) according to the manufacturer's instructions. cDNA was synthesized by using TransScript One- Step gDNA Removal and cDNA Synthesis SuperMix (TRAN).
To assess mRNA expression, qRT-PCR was conducted using SYBR Premix Ex Taq™ II (TaKaRa, Japan). The target gene ex-

| Statistical analysis
All experiments were performed three times, and the results were shown as mean ± SD. To compare the statistical differences, GraphPad Prism software was used by two-tailed Student's t test or one-way ANOVA as approximate. P value less than 0.05 was considered significant.

| Melatonin and dabrafenib synergized to inhibit the proliferation of anaplastic thyroid cancer cells
To study the role of melatonin in dabrafenib-mediated cell prolifera- Conclusively, melatonin combined with dabrafenib has a synergistic inhibitory effect on the viability of ATC cells.
We then assessed the effect of combination treatment on the colony formation abilities of ATC cells. As shown in Figure 1C and D, compared to single agents, the combination of melatonin and dabrafenib significantly increased the inhibition of colony formation in SW1736 and OCUT1 cells.
In addition, we next explored the potential molecular mechanisms by which combination of melatonin and dabrafenib synergistically inhibited the proliferation of ATC cells. The results showed that the expression of phosphorylated AKT protein involved in proliferation was significantly reduced after treatment with melatonin and dabrafenib in SW1736 and OCUT1 cells ( Figure 1E). Taken together, these data indicated that the combination of melatonin and dabrafenib has a synergistic effect in inhibiting thyroid cancer cells proliferation by targeting AKT signalling.

| Combination of melatonin and dabrafenib synergistically induced cell cycle arrest
To evaluate whether the synergistic inhibition of melatonin and dabrafenib on ATC cells growth was related to cell cycle arrest, SW1736 and OCUT1 cells were treated with melatonin or dabrafenib alone or together for 48 hours, followed by cell cycle analysis. As shown in Figure 2A

| Melatonin synergized with dabrafenib to induce apoptosis in anaplastic thyroid cancer cells
To

| Melatonin and dabrafenib had synergistic inhibitory effects on the migration and invasion of anaplastic thyroid cancer cells
We also evaluated the synergistic regulation of melatonin and dabrafenib on the migration and invasion abilities of ATC cells by performing scratch assays and transwell invasion assays, respectively. As shown in Figure 4A and B, monotherapy with melatonin or dabrafenib alone inhibited cell migration, but the combination of these two agents significantly enhanced the inhibitory effect of cell migration. Consistent with the inhibition of cell migration, the combinational use of melatonin and dabrafenib also led to a significant reduction in the invasiveness of ATC cells compared to single agents ( Figure 4C and D).

| Melatonin synergistically enhanced dabrafenib-induced growth inhibition of anaplastic thyroid cancer cells by down-regulating hTERT signalling
We next explored the molecular events that may be involved in the growth inhibition of ATC cells mediated by co-treatment with melatonin and dabrafenib. hTERT, a major component of telomerase, has been shown to contribute to transforming normal human cells into cancer cells. Studies have suggested that inhibition of hTERT significantly reduced the proliferation, invasion and migration abilities of ATC cells. 31 To determine whether combination treatment with melatonin and dabrafenib could affect hTERT signalling in ATC cells, we treated melatonin or dabrafenib alone or together, and examined the expression of hTERT. We found that co-treatment with melatonin and dabrafenib significantly reduced the expression of hTERT at protein and mRNA levels ( Figure 5A and B).
To further identify hTERT expression was involved in the synergistic growth inhibition of melatonin and dabrafenib in ATC cells, SW1736 and OCUT1 cells were transfected with hTERT control vector or overexpression plasmids after pre-treatment with melatonin and dabrafenib. After 48 hours of treatment, the expression of hTERT protein, cell viability and cell cycle arrest was determined.
The results showed that the expression of hTERT was significantly increased in both cell lines ( Figure 5C and D). In addition, the decreased cell viability caused by the combination of melatonin and dabrafenib was reversed by hTERT overexpression ( Figure 5E).
Similarly, overexpression of hTERT significantly inhibited the induction of cell cycle arrest at G1-phase mediated by combination treatment ( Figure 5F and G). Overexpression of hTERT also increased the phosphorylation of AKT and significantly promoted the expression of CDK2 and cyclin D1. These results showed that the enhanced inhibitory function of melatonin and dabrafenib on ATC cells growth is at least partially achieved by inhibiting hTERT signalling pathway.

| D ISCUSS I ON
In the past few years, most thyroid cancer treatments have targeted known oncogenic mutations, such as BRAFV600E, to convert the progression of thyroid cancer. In view of the high frequency of BRAFV600E mutations in ATC, the use of selective BRAFV600E inhibitors was a good choice for improving therapeutic specificity The main cause of death in ATC patients is related to its profound metastasis and invasion characteristics 38  hTERT is a major component of human telomerase that prolongs the end of chromosomes and maintains chromosomal stability, resulting in cellular immortalization and tumorigenesis. 39 For a long time, studies on hTERT were mainly focused on its ability to maintain telomere length to continuously promote cell proliferation. However, in recent years, hTERT has also been found to have non-telomere-dependent functions, 40 such as regulation of gene expression, cellular signalling pathways, cell cycle, protection of mitochondrial DNA and regulation of DNA damage response. [41][42][43][44][45] It was usually overexpressed in a variety of tumours, including thyroid cancer. 31 Accumulating evidence has suggested that overexpression of hTERT was not only related to the aggressive behaviours of thyroid cancer cells, 46 but also predicted the early recurrence of thyroid cancer patients. 47 Knockdown of hTERT resulted in decreased proliferation, invasion and migration abilities of ATC cells. 31 Moreover, hTERT was found to induce thyroid cancer cell proliferation by regulating PTEN/AKT signalling pathway. 48 Considering that melatonin exerted its anti-tumour effects by inhibiting the expression of hTERT and the activity of telomerase, 49

| CON CLUS IONS
In summary, our results suggested that melatonin and dabrafenib synergistically inhibited the growth of anaplastic thyroid cancer cells, as evidenced by synergistic inhibition of proliferation, migration and invasion, and synergistic enhancement of cell cycle arrest and apoptosis induction. In addition, we also investigated the molecular mechanisms that may be involved in this combination therapy, and found that melatonin synergized the anti-tumour effect of dabrafenib by inhibiting hTERT signalling in human anaplastic thyroid cancer cells ( Figure 6). All these data indicated that the combinational use of melatonin and BRAF-targeting agent dabrafenib has the potential to become a novel approach in the treatment of anaplastic thyroid cancer.

ACK N OWLED G EM ENTS
This work was supported by the funds from the National Natural Science Foundation of China (No. 81702831 to Ranran Tang and No. 81703100 to Jing Cao).

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.