Low dose radiation regulates BRAF-induced thyroid cellular dysfunction and transformation
The existence of differentiated thyroid cells is critical to respond radioactive iodide treatment strategy in thyroid cancer, and loss of the differentiated phenotype is a trademark of iodide-refractive thyroid disease. While high-dose therapy has been beneficial to several cancer patients, many studies have indicated this clinical benefit was limited to patients having BRAF mutation. BRAF-targeted paired box gene-8 (PAX8), a thyroid-specific transcription factor, generally dysregulated in BRAF-mutated thyroid cancer.
In this study, thyroid iodine-metabolizing gene levels were detected in BRAF-transformed thyroid cells after low and high dose of ionizing radiation. Also, an mRNA-targeted approach was used to figure out the underlying mechanism of low (0.01Gyx10 or 0.1Gy) and high (2Gy) radiation function on thyroid cancer cells after BRAFV600E mutation.
Low dose radiation (LDR)-induced PAX8 upregulation restores not only BRAF-suppressive sodium/iodide symporter (NIS) expression, one of the major protein necessary for iodine uptake in healthy thyroid, on plasma membrane but also regulate other thyroid metabolizing genes levels. Importantly, LDR-induced PAX8 results in decreased cellular transformation in BRAF-mutated thyroid cells.
The present findings provide evidence that LDR-induced PAX8 acts as an important regulator for suppression of thyroid carcinogenesis through novel STAT3/miR-330-5p pathway in thyroid cancers.
KeywordsLow dose radiation, LDR Thyroid cancer Paired-box domain 8, PAX8 miR-330-5p Thyroglobulin, TG
- BRAF V600E
Amino acid substitution at position 600 in BRAF from a valine (V) to a glutamic acid (E)
Serine/threonine-protein kinase B-Raf
Enzyme-linked immunosorbent assay
Fluorescence-activated cell sorting
Janus kinase 2
Mitogen-activated protein kinase kinase-Extracellular regulated kinases
Suppressor of cytokine signaling proteins
Signal transducer and activator of transcription 3
Recently activating somatic mutations in the BRAF proto-oncogene has been discovered in various malignancies, such as in melanoma (60–70% of cases) [1, 2], colon cancer (10%) [3, 4] including thyroid cancer (35–70%) [5, 6]. Thyroid tumors are the most frequent neoplasms of the endocrine system . Well-differentiated thyroid carcinomas account for > 85% of all thyroid cancers including papillary and follicular carcinomas. In early 2003, BRAF mutations were reported in thyroid cancer with an occurrence ranging from 25 to 42% . Papillary thyroid carcinoma (PTC) and anaplastic thyroid carcinoma (ATC) have only been reported with these mutations , however it has not been identified in follicular thyroid carcinoma (FTC), or benign thyroid adenomas . Among all thyroid subtypes, PTC is the most prevalent and have an aggressive behavior , while undifferentiated anaplastic thyroid carcinoma accounts for 3 to 5% of all thyroid cancers .
BRAF is a serine/threonine kinases belongs to the RAF family. RAF proteins are part of the RAF-MEK-ERK pathway [mitogen activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase], a prominently conserved signaling component in eukaryotes. Once activated through binding to RAS in its GTP-bound state, RAF kinases phosphorylate MEK, thereby phosphorylates and activates ERK . Activation of BRAF has originated as the most prevalent oncogenic mutation in thyroid carcinoma [5, 6, 13]. A trans-version from thymine to adenine (T1799A), leading to a Glu for Val substitution at residue 600 (V600E), accounts for > 92% of BRAF mutations in thyroid carcinomas . Consistent with a pivotal role in thyroid cancer initiation, BRAFV600E mutation has been identified in microcarcinomas , and it was revealed to induce transformed features in thyroid follicular cells in culture conditions . A recent report suggests that ultraviolet radiation accelerates BRAF-driven melanogenesis by targeting TP53 . Given that above well-characterized role of BRAF mutations prompted us to explore the functions of low dose of ionizing radiation (LDR) in BRAF-mediated cellular transformation in thyroid cancer cells. The advance of interventional radiology has attracted growing interest in the biological effect of LDR below 0.1Gy doses . From our previous studies, it has become apparent that the LDR has the potential to block KRAS-driven cellular transformation  and metastatic cancer progression in breast cancer cells . As, most of the thyroid patients can be cured with surgical treatment together with radioactive iodide, however BRAF-mutated thyroid cancer cells have lower expression of sodium/iodide symporter (NIS), thyroid transcription factors (TTF-1 and TTF-2), and thyroglobulin (TG) than those cells having wild-type (WT) BRAF , and are particularly refractory to radioiodine therapy . The ability of differentiated thyroid cells to accumulate iodide is clinically highly relevant as it makes feasible for thyroid cancer patients to be treated with ablative doses of radioactive iodide after stimulation by thyroid-stimulating hormone . Thus, the maintenance of the thyroid differentiated phenotype during tumor transformation has a critical impact in thyroid cancer patient’s survival . PAX8, a member of the paired box (PAX) family of transcription factors are required for the maintenance of the thyroid differentiated phenotype . Along with the other thyroid transcription factors TTF-2 and TTF-1, PAX8 is intricate in development of thyroid follicular cells as well as expression of thyroid-specific genes such as the NIS, and TG [24, 25]. These genes are essential for thyroid differentiation as they mediate the metabolism of iodide, leading to the synthesis of active thyroid hormone. One of the most important and well-established transcriptional targets of PAX8 is NIS. This symporter is a central plasma membrane protein that mediates active iodide transport in the thyroid and other tissues . In the healthy thyroid cells, NIS-mediated iodide uptake is the first step in thyroid hormone biosynthesis. Therefore, loss of the thyroid differentiated phenotype, particularly loss of NIS function, is one of the most important hallmarks of thyroid cancer progression.
The purpose of this study was to compare the iodine metabolizing genes expression profile induced by expression of BRAFV600E in thyroid cells using LDR. Given that several miRNAs were associated with less differentiated tumors , here, we took a unified approach based on the existence of the miRNAs for LDR influence in thyroid carcinoma. Here, we investigated the restorability of thyroid specific genes expression by suppression of the miR-330-5p in PTC/ATC after LDR exposure. These findings could provide the positive role of LDR in thyroid cells expressing BRAF-mutant and to characterize those miRNAs involved in the alteration of genes essential for thyroid differentiation, namely PAX8.
Materials and methods
Chemicals and antibodies
Antibodies specific to pJAK1(Tyr1022/1023), JAK1, pJAK2(Tyr1007/1008), JAK2, STAT3 and BRAF were obtained from Santa Cruz Biotechnology, Inc. Antibodies to pSTAT3 (Tyr705) were purchased from Cell Signaling Technology. PAX8 antibody was purchased from Abcam (Seoul, Korea). NIS antibody was obtained from GeneTex, Inc., whereas 4,6-Diamidino-2-phenylindole (DAPI) and β-actin were obtained from Sigma, Korea. Anti-mouse or rabbit Alexa Fluor 488 and anti-rabbit or mouse Alexa Fluor 546 were purchased from Invitrogen. All controls, silencing RNA, micro RNAs mimics and inhibitor were bought from Genolution Pharmaceuticals, Inc., Korea. All primers including control and microRNA primers were designed and purchased from Macrogen, Korea. Hanks’ Balanced Salt solution (HBSS), HEPES, Ammonium cerium (IV) sulphate, Sodium arsenite (III) and sodium iodide were purchased from Sigma-Aldrich, Korea. The uptake buffer consisted of HBSS supplemented with HEPES (10 mM final conc.) was freshly prepared prior to assay.
Cell culture and transfection
Human thyroid normal follicular N-thy ori-3-1, BHP 10–3, SNU-80, BCPAP, 850-5C and SNU-790 thyroid cancer cell lines were kindly gifted by Dr. Min-Jung Kim (KIRAMS, Korea). 850-5C cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose (Hyclone, Korea) whereas other cell lines were cultured in Roswell park memorial institute medium (RPMI-1640). Normal thyroid cells were supplemented with 20 mM glutamine (Gibco, Korea). All cell cultures were supplemented with 10% fetal bovine serum (Hyclone) and maintained at 37 °C in a 5% CO2 atmosphere. Cells were passaged every two-three day and often treated with plasmocin™ (Invivogen) to prevent mycoplasma contamination. We used all cell lines about 25–30 passages to perform our experiments. Cell transfection was accomplished in 75–85% confluent cells using Lipofectamine 2000 Reagent (Invitrogen, USA) according to the manufacturer’s protocol. BRAFV600E virus supernatant was made by Min-Jung Kim (KIRAMS, Korea) and treated directly to normal thyroid cells at a confluency of 75–80%.
Thyroid cells were plated in 60-mm cell culture dishes and irradiated with a 137Cs laboratory γ-irradiator (LDI-KCCH 137, Seoul, Korea) using same procedure as described in our report previously .
Western blot analysis
For Western blotting analysis, cells were harvested and lysed for protein extraction using cell lysis buffer [40 mM Tris-HCl (pH 8.0), 120 mM NaCl, 0.1% Nonidet-P40] supplemented with protease inhibitors. Proteins samples were separated by SDS-PAGE and blotted onto nitrocellulose membranes (Amersham, IL), blocked in 5% skim milk for 1 h (h) at room temperature and incubated with the primary antibodies overnight at 4 °C: anti-BRAF (1:1000), and anti-β-actin (1:1000). The blots were developed using horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h and visualized using enhanced chemiluminescence (ECL) procedures.
Real time PCR analysis
Cell samples RNA was extracted using the Trizol reagent (Ambion). Quantification of RNA was done using the NanoDrop™ Spectrophotometer according to the manufacturer’s protocol. All qRT-PCR reactions were determined using the KAPA SYBR FAST qPCR kit from KAPA Biosystems (Wilmington, MA, USA). Amplification reactions were carried out in a Rotor Gene Q (Qiagen, Korea), and results were expressed as the fold change calculated by the ΔΔCt method relative to the control sample. β-actin was used for normalization as a control. For miRNA analysis, U6 small nuclear RNA was used as a control to determine relative miRNA expression.
Tissue samples and immunohistochemistry
Paraffin blocks and slides from 10 cases of PTC and 4 cases of normal (between 1999 and 2013) were recovered from the archives of the Korea Cancer Center Hospital. Tissue sections were deparaffinized, rehydrated, exposed to antigen retrieval in antigen retrieval buffer, incubated with 3% hydrogen peroxide and non-specific binding was blocked using bovine serum albumin. These sections were incubated with mouse monoclonal anti-PAX8 antibody (1:400; Abcam) overnight at 4 °C, and then incubated with biotinylated secondary antibody bound to a streptavidin–horseradish peroxidase complex. The bound antibody was distinguished using 3,3-diaminobenzidine and the sections were counterstained with hematoxylin, dehydrated and mounted. All sections were recorded by individual pathologists.
For immunofluorescence, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Afterwards, cells were incubated with the PAX8 (anti-mouse, 1:200), NIS (anti-rabbit, 1:200) primary antibodies in a solution of PBS with 1% bovine serum albumin and 0.1% Triton X-100 at 4 °C overnight. Next day, staining was visualized using anti-rabbit or anti-mouse Alexa Flour 488 and anti-rabbit or anti-mouse Alexa Flour 546 antibodies, and cells nuclei were counterstained using 4,6-diamidino-2-phenylindole (DAPI; Sigma). Stained cells were imaged with a confocal fluorescence microscope (Nikon).
To examine the PAX8 positive cell population with respected experiments, the LDR-exposed cells were fixed with 2% paraformaldehyde and permeabilized with 90% methanol as suggested by manufacturer’s protocol. Then, these cells were labeled with an anti-PAX8 antibody. A respective control was also prepared and tested for each sample. The percentage of PAX8 positive cells were determined using PE (Alexa-flour488) and was analyzed using a BD FACSVerse cytometer and the FACS suite software.
Thyroid stimulating hormone receptor (TSHR) ELISA assay
For TSHR determination, cell culture supernatant/blood serum was collected as desired time points in each experiment using colorimetric TSH receptor ELISA kit (LS biosciences, LS-F12873). All assay steps were performed according to kit manual instructions. This assay utilizes an antibody specific for Human TSH coated on a 96-well plate. The intensity of the color is measured at 450 nm using an ELISA plate reader.
In vitro iodine uptake assay
Briefly, three days after cell seeding in 96-well cell culture plate, BCPAP cells formed approximately 75–80% confluency and assay procedure was adopted from previous report .
Soft agar and sphere formation assays
For cellular transformation studies, we performed soft agar colony assays and sphere forming assays. To observe anchorage independent growth, a cell suspension (2 × 104 cells) was suspended in 0.4% agar in growth medium and seeded in triplicate in 60-mm dishes pre-coated with 0.8% agar in growth medium and incubated at 37 °C with 5% CO2. After 16 days, colonies were photographed and counted in five randomly chosen fields and expressed as means of representative of two independent experiments. For tumor sphere assays, cells were plated as single-cell suspension in ultralow attachment 6-well plates and grown in serum free DMEM/F12 medium supplemented with 20 μl ml− 1 B27 (Invitrogen), 20 ng ml− 1 EGF and 20 ng ml− 1 bFGF. Fresh media (300 μl) was added every 3 days. At day 7, tumor spheres were counted and photographed. All sphere and colony formation assays were performed in triplicate.
To make an orthotopic model of thyroid carcinoma, NSG male mice (8–10 weeks old) Jackson Laboratories, Bar Harbor, ME, USA) were used according to previously established method . Housing and all experimental animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Center for Laboratory Animal Sciences, the Medical Research Coordinating Center, and the HYU Industry-University Cooperation Foundation. We used stable N-thy ori-3-1 thyroid cells (Mock and BRAF-untreated and LDR-treated) for animal experiment and 2.5 × 105 cells/mouse (right side of thyroid gland) times the number mice injecting. Mice were sacrificed 25 days after cell injection and serum plasma were collected for TSHR Elisa assay. Injected thyroid glands were also excised for further experiments.
All experimental results are represented as the mean ± standard deviation (S.D.) of at least three independent tests. The statistical analysis was performed using the parametric Student’s t-test to check the significance levels. Levels of significance are indicated as *p < 0.05; **p < 0.01; and ***p < 0.001.
BRAF mutation induces hypothyroidism in thyroid cancer
LDR inhibits BRAF-driven cellular transformation and rescues repression of iodine-metabolizing gene expression responsible for thyroid cancer initiation
PAX8 upregulation is critical in decreased cellular carcinogenesis
Loss of miR-330-5p leads to PAX8 upregulation by LDR and improve NIS/TG expression
STAT3 acts as an upstream regulator for miR-330-5p essential for thyroid malfunction decreased by LDR
Expression of thyroid metabolizing genes are dramatically restored in BRAF-mutant mice model
Generally, the diagnosis for PTCs is suitable unless the tumor presents a well or poorly differentiated phenotype. It is believed that dedifferentiation is correlated with molecular alterations of proteins that permit the thyrocytes to concentrate the iodine, which render the tumor refractive to radioiodine treatment . Aberrant activation of BRAFV600E mutation is accompanying with the loss of radioiodine uptake and therapy failure in PTC. Reliably, BRAFV600E mutation is highly predominant (80–90%) in recurrent radioiodine-refractory PTC/ATC patients [42, 43], as comparison with the lower occurrence of BRAFV600E mutation (40%) in primary PTC . Several studies have demonstrated an association of BRAFV600E mutation with reduced expression of thyroid iodide-metabolizing genes namely NIS, TSHR, TG in thyroid cancer and induces hypothyroidism [19, 44]. Moreover, Liu et al. showed that BRAFV600E is directly involved in impairment of the expression of almost these genes using a MEK inhibitor . Consistent with this studies, we also identified that PAX8 has lower expression in PTC and ATC patients having BRAFV600E mutation (Fig. 1g).
Molecular events leading to the loss of thyroid differentiation in thyroid cancer draws much attention, especially loss of NIS expression. NIS is required for the active transport of iodide into the thyroid cells, and for the diagnosis and therapeutic management of thyroid cancer patients treated with radioiodine. We determine for the first time that LDR critically enhanced PAX8 expression levels in PTC and ATC expressing BRAFV600E mutant. PAX8 is a transcription factors that controls the transcription of NIS, is essential for the thyroid gland development and for maintaining the differentiated state in the normal thyroid gland . We also show that PAX8 modulated NIS expression in thyroid cancer after LDR exposure in thyroid cells. PAX8 overexpression facilitate NIS expression on plasma membrane and improve its localization (data not shown). To figure out how the LDR-mediated induction of PAX8 involved in thyroid-specific activation of the NIS and TG gene, we took advantage of the miRNA-based approach. Interestingly, the results obtained with the prediction online tools indicate that the miR-144-3p and miR-330-5p containing the already known PAX8 targeting sites could be involved in LDR function. In real time PCR assays, LDR treatment downregulated both miRNAs expression very efficiently, whereas the HDR responded poorly to these miRNAs, showing an activation. On the contrary, miR-330-5p mimetics interfered with the effect on LDR on NIS, TG and colony formation in PTC (Figs. 4i-j). As miRNAs expression decreases in response to LDR rather than HDR, we identified upstream molecule which can be affected through LDR. The results emphasize that JAK2/STAT3 activation was harbored after LDR in PTC/ATC (Fig. 5a). The STAT3 overexpression state contradicted LDR function in maintenance of PAX8, NIS, TG gene expression that may contribute to cellular transformation (Figs. 5c & f). Despite the sensitivity of LDR to BRAF-induced cellular transformation in thyroid cells, their BRAF expression levels were not downregulated by LDR from those detected in BRAFV600E cells. It could be assumed that some other pathway is intricate in STAT3 activation rather than BRAF phosphorylation. To resolve this question, we measured the SOCS family proteins, whose expression negatively regulate JAK/STAT signaling after LDR treatment. In doing so, we found that SOCS3 and SOCS4 were majorly induced in LDR-treated PTC cells (Additional file 1: Figure S3D). In addition, Kleiman et al., suggested that the marked reduction of TSHR co-operates with oncogenic BRAF induced dysregulation of thyroid development  and TSHR signaling is required for expression of a subset of thyroid-specific genes during development . Our data indicated LDR effectively recovered TSHR levels in thyroid carcinoma cells (Additional file 1: Figure S1B), perhaps decreasing STAT3-miRNA axis route accounting for the high undifferentiated phenotype.
We thank Prof. Hee-Yong Chung (Department of Medicine, Hanyang University, Korea) for providing pCMV6 empty and pCMV6-STAT3 vectors.
This work was supported by the Ministry of trade; industry & energy grant No. 20131610101840 and also by grant of the Korea Institute of Radiological and Medical Sciences (KIRAMS), funded by Ministry of Science and ICT (MIST), Republic of Korea (50535-2019).
Availability of data and materials
Supporting data could be requested to corresponding or first author anytime during manuscript procession.
NK conceived and performed the cell-culture experiments, analyzed the data; NK, NKK, MJK and SJL finalized the figure format and wrote the manuscript. NK and MYC contributed to the animal experimental design and performed in vivo experiments. JHK contributed to graphical abstract; HJC, JKM, contributed to the data analyses paper discussion; SJL, CSK, and SYN conceived, directed the research. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Human patient samples were recovered from the archives of the Department of Pathology at the Korea Cancer Center Hospital. The consent medical records of the patients corresponding to these cases were reviewed. This study was permitted by the Institutional Review Board of the Korea Cancer Center Hospital. In case of mice studies, animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Center for Laboratory Animal Sciences, the Medical Research Coordinating Center, and the HYU Industry-University Cooperation Foundation.
Consent for publication
On the behalf of all co-authors, I provide the consent for publication of this manuscript.
The authors declare that they have no competing interests.
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