Bisphenol A at a human exposed level can promote epithelial‐mesenchymal transition in papillary thyroid carcinoma harbouring BRAF V600E mutation

Abstract Bisphenol A (BPA), a ubiquitous endocrine‐disrupting chemical, alters the function of endocrine system and enhances the susceptibility to tumorigenesis in several hormone‐dependent tumours as thyroid carcinoma. About 50% of papillary thyroid cancers (PTC), the most common type of thyroid malignancy, harbours the BRAF V600E mutation. This study aimed to investigate a potential combined effect of BPA exposure and BRAF V600E mutation on epithelial‐mesenchymal transition (EMT) in PTC. Firstly, the level of BPA in plasma, the evaluation of BRAFV600E mutation and the level of EMT‐related proteins in PTC samples were individually determined. Additionally, the migration, invasion, colony formation capacity and the expression of EMT‐related proteins after exposure to BPA were precisely analysed in vitro thyroid cells genetically modified by the introduction of BRAF V600E mutation. Moreover, ERK‐Cox2 signalling pathway was also introduced to explore the possible mechanism in PTC development. As expected, whether the clinical investigation or cultured thyroid cells demonstrated that BPA at a concentration compatible with human exposed levels (10‐7 M) synergized with the BRAF V600E mutation promoted EMT via the activation of ERK‐Cox2 signalling pathway. Our findings offer some evidence that BPA as an environmental risk factor can facilitate the progression of PTC harbouring BRAF V600E mutation.


| INTRODUC TI ON
Thyroid carcinoma has been appreciably increasing worldwide in recent decades, which gradually becomes the most common solid malignant tumour of the endocrine system and is highly prevalent in middle-aged people. 1 Generally speaking, papillary thyroid carcinoma (PTC), the most common thyroid malignancy, accounts for over 80% of all cases and is usually associated with favourable survival. 2 However, recurrence, metastases and cancer death may occur in 10%-15% of patients with more aggressive types, such as epithelial cell-derived PTC. 3 Therefore, studies on the detailed mechanisms underlying PTC metastasis is of significance in improving our understanding of the pathogenesis process and identifying valid, targeted treatments.
Epithelial-mesenchymal transition (EMT) is a biological process that epithelial cells lose their junctions and apical-basal polarity, and acquire migratory and invasive capabilities. 4 In the context of neoplasias, EMT confers on cancer cells increased tumour-initiating and metastatic potential, 5 in particular, epithelial-derived malignant cells. 6 The cells consequently acquire a spindle-shaped mesenchymal morphology and express markers associated with the mesenchymal cell state, notably neural cadherin (N-cadherin) and matrix metallo proteinase-9 (MMP-9), while the expression of E-cadherin and certain cytokeratins is typically lost. 7,8 Genetic abnormalities and epigenetic alterations have been recognized as a crucial driver in EMT. Remarkably, BRAF V600E mutation, the transformation of valine (V) to glutamic acid (E) at amino acid 600 (pV600E) resulting from T-to-A (c1799T > A) substitution at nucleotide 1799, has been identified as a targetable, oncogenic mutation in many cancers, present in some 30%-70% of PTC with pronounced transcriptomic and prognostic consequence. 9 Mechanistically, the mutation constitutively activates the BRAF kinase in the MAPK pathway to promote aggressiveness and contribute to a worse prognosis, advanced stage, lymph node metastasis as well as the resistance to traditional radiation therapy of PTC.
Bisphenol A (BPA) as a synthetic ubiquitous endocrine-disrupting chemical (EDC) is used greatly in the manufacture of plastic products. [10][11][12] The growing epidemiological studies indicate that BPA has been associated mainly with hormone-sensitive cancers including thyroid cancers as it interacts with oestrogen receptors and acts as an agonist or antagonist via oestrogen receptor-dependent signalling pathways. For example, Zhou Z's study indicated that the BPA level in urine of PTC patients was higher than the healthy control, which reminded that BPA exposure may potentially promote the pathogenesis and progression of PTC 12 ; Soto's studies suggested the increasing incidence of thyroid carcinoma was associated with BPA exposure in almost all age groups. 13 Recent evidence has highlighted that BPA has an increasingly crucial role in EMT. For instance, Zhai's study indicated that BPA regulated snail-mediated EMT in hemangioma cells, consistently with Oral's investigation in ovarian cells and Kim's research in human breast cancer cells MCF-7 CV. [14][15][16] Despite inroads that have been made in identifying BRAF V600E mutation's potential role in the EMT process of PTC, the joint effect of BPA exposure on EMT induced by BRAF V600E mutation remains uncertain. Accordingly, a clinical investigation of PTC patients and an in vitro genetically emerging thyroid cell model was performed in the present study to assess whether BPA exposure especially at a human exposed level combined with BRAF V600E mutation could promote the EMT process and further enhance migration and invasion of thyroid cells. Moreover, ERK-Cox2 as an activated signalling pathway was also introduced to explore the possible mechanism of thyroid carcinogenesis. Hopefully, our current study may contribute to understanding scientific significance for early prevention of thyroid carcinoma related to endocrine-disrupting chemicals exposure.

| Materials pre-treatment
In order to reduce the interference of environmental BPA pollution in our experiment, all the consumables (including blood collection tubes, containers and pipettes) were made of glass products in the subject process. The washed glassware was first of all soaked in potassium dichromate solution for 24 hours, afterwards rinsed with ultrapure water and then rinsed with 75% ethanol solution, at last, dried in a dry box before use. The plastic consumables used in vitro were made of general purpose polystyrene without BPA.

| Detection of BPA level in plasma
Human BPA ELISA kit, purchased from Shanghai Enzyme-linked Biotechnology Company, was used to measure BPA content in plasma of the study subjects. Used suited EDTA as an anticoagulant, mixed 15 minute, centrifugated 20 minute at the speed of 1000 g and collected supernatant. Then, the experimental procedure and formulas for the calculation strictly followed the kit instructions.

| Evaluation of BRAF V600E mutation in tumour
Immunohistochemistry was performed as previously described to detect the BRAF V600E mutation. 17 PTC tissue samples were sliced into 4-μm sections and incubated overnight at 4°C with the addition of a commercially available BRAF V600E mutation-specific antibody (dilution 1:100, clone VE1; Spring Bioscience, Pleasanton, CA, USA). Further, the sections were incubated with biotin-labelled secondary goat anti-rabbit immunoglobulin G (IgG) at 37°C for 20 minutes, stained with diaminobenzidine and counterstained with haematoxylin. The negative controls were performed by omitting the primary antibody. The BRAF V600E mutation was evaluated by two experienced pathologists (DES) with the blind method. Diffuse homogeneous cytoplasmic staining in cells was considered as a positive BRAF V600E mutation, while non-specific staining of colloids and equivocally weak or focal cytoplasmic staining was considered as BRAF V600E wild-type.

| Immunohistochemical staining
The procedure of immunohistochemical staining for E-cadherin, N-cadherin and MMP-9 was the same as the previous description.
Finally, the slides were scanned and images were captured in 5 randomly selected visual fields for analysis under a Digital Pathology Scanner (Aperio CS2, Leica Biosystems, USA).

| Plasmids transfection
The pIRESpuro3-EGFP-Flag-6 × His-BRAF-T1799A plasmid and the control plasmid pIRESpuro3-EGFP-Flag-6 × His (OBio, Shanghai, China) were transfected by Lipo8000™ reagent (Beyotime, China) according to the manufacturer's instructions. After 48 hours transfection, the medium was replaced with different concentrations of BPA-containing complete medium and the cells were further detected for the following biological function tests.

| Preparation of BPA-containing culture medium
BPA (Chengdu Aikeda Chemical Reagent Co., Ltd., China) was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, USA) to form a 1 M stock solution stored at −20°C. The stock solution was diluted with complete medium to obtain a drug-containing culture medium with a final BPA concentration of 10 -3 to 10 -10 M for cell treatment.
The control group was treated with a complete culture medium containing 0.1% DMSO.

| Cell proliferation assay
Cells were seeded in 96-well plates at a density of 3000 cells per well. After 24 hours incubation, the cells were treated with different concentrations of BPA for 24, 48 or 72 hours. Then 20 μL MTS was added and cells were incubated for 2h to measure the cell viability with a microplate reader (EON, BioTek, USA).

| Scratch wound assay
Cells were seeded in a 6-well plate and transfected when the cell fusion rate reached 70%-80%. After 48 hours of cell transfection, scratches were made. After treatment with BPA at different concentration, the wound healing of cells at the same location was observed with a digital microscope (EVOS XL Core, Thermo Fisher Scientific, USA) and the migrated rate of the scratch wound was evaluated using Image J software (National Institutes of Health, USA).

| Cell migration and invasion assays
Transwell chambers were put into 24-well plates, and the apical chamber of the basement membrane was coated with Matrigel diluent (Corning, USA). The cells (3 × 10 4 cells/insert for migration assay; 5.5 × 10 4 cells/insert for invasion assay), transfected different plasmids for 48h, were suspended in 150 μL of serumfree medium and seeded onto the upper chamber, while the lower chamber was loaded with 600 μL complete medium containing different concentrations of BPA. After 24 hours of incubation, the non-migrating or non-invading cells were removed from the upper surface of the membrane by gently scrubbing with the cotton-tipped swab. Cells on the lower surface of the membrane were fixed with methanol and then stained with 0.2% crystal violet followed by two washes with PBS. Thereafter, cells from five randomly selected microscopic fields were photographed and counted (EVOS XL Core, Thermo Fisher Scientific, USA).

| Colony formation assay
Cells were incubated in 6-well plates for 12 hours, then transfected with the corresponding vectors for 48 hours. Afterwards, 2000 cells per well were seeded in 12-well plates and exposed to different concentrations of BPA. After 7 days, colonies were fixed with 4% paraformaldehyde and stained using 0.2% crystal violet. Finally, colony numbers were counted and analysed using the light microscope (Model 200-BFFL-S, Nexcelom Bioscience, USA).

| Total RNA extraction and quantitative realtime PCR
Total RNA was extracted from thyroid carcinoma tissues with Trizol reagent (Invitrogen Inc, Burlington, ON, Canada). One microgram of total RNA was converted to cDNA and then was amplified in duplicate using SYBR Premix Ex Taq II (Takara, Dalian, China) and a Light Cycler 480 II (Roche, Switzerland) PCR detection system. The relative quantification of mRNA levels was calculated by the ln2 −ΔCt method, normalized to GAPDH. The primer sequences are shown in Table S1.

| Statistical analysis
Data statistical analysis was carried out using the SPSS 20.0 software. The enumeration data were expressed in terms of frequency and percentage and measurement data was expressed as mean ± standard deviation (SD). Comparisons between two groups were determined by Student's t test. Multiple-group comparisons were performed by one-way ANOVA and LSD multiple comparison tests. Each independent experiment performed in triplicate and represented as 'n = 3', and P < .05 was considered as statistically significant.

| Quantitation of BPA exposure in plasma
The demographic data of study subjects were present in supplementary materials (Table S2). BPA inter exposure in plasma of study subjects was at 10 -7 M, a common human exposed level, while no significant difference was found between healthy controls and PTC patients. Subsequently, stratified analysis by age, gender, smoking habits and alcohol drinking status demonstrated no statistical difference as well (Table 1).

| BRAF V600E mutation in PTC tissue
According to the result of immunohistochemical staining with the anti-BRAF V600E VE1 in tumour tissues from 45 PTC patients, 57.8% of the patients (26/45) were concluded to harbour the BRAF V600E mutation.
In detail, the diffused brownish yellow granules in the cytoplasm represented for typical positive BRAF V600E mutation ( Figure S1).

| BRAF V600E mutation promoted EMT in PTC
The IHC-P Scores (IHS) of EMT-related proteins were shown in Figure 1. We found that the epithelial cell marker E-cadherin expression in BRAF V600E mutant tissues was significantly repressed compared with the wild-type ones ( Figure 1B), whereas N-cadherin and MMP-9 protein expression was significantly up-regulated in BRAF V600E mutated samples ( Figure 1E and H). Consistently, transcriptional repression of E-cadherin but activation of N-cadherin and MMP-9 was observed in BRAF V600E mutated samples ( Figure 1C, F  (Figure 2A), while N-cadherin ( Figure 2B) and MMP-9 ( Figure 2C) were increased in PTC patients with BRAF V600E mutation compared with wild-type ones. Meanwhile, in the BPA high-exposure group, the above proteins showed a similar trend to BPA low exposure. Remarkably, E-cadherin mRNA expression showed significant repression in ones harbouring BRAF V600E mutation combined exposed to high BPA concentration, but such a trend was not observed in the low BPA exposure group ( Figure 2D); Meanwhile, high BPA concentration might induce the repression of E-cadherin but the activation of N-cadherin and MMP-9 protein expression in BRAF V600E mutated tissues in spite of no statistical significance (Figure 2A-2C). The above results indicated that BPA exposure might promote EMT in PTC harbouring BRAF V600E mutation.

| Establishment of the BRAF V600E overexpressed cell model
The transfection efficiency in the present experiment was up to 60%-75% after 48h's transfection ( Figure 3A). Subsequently, we confirmed that compared with the parental and empty vector-transfected Nthy-ori 3-1 cell, the introduction of BRAF V600E -mutated vector significantly increased the expression of BRAF ( Figure 3B). In addition, activation of BRAF-ERK signalling pathway in Nthy-BRAF V600E cells ( Figure 3C), which was evidenced by the increased protein levels of BRAF and p-ERK, indicated that the BRAF V600E overexpressed thyroid cell model was successfully established. Remarkably, Nthy-BRAF V600E cells had a spindle transformed shape other than the Nthy-Vector cell after 48h's transfection, as shown in Figure 3D-3F.

| BPA exposure augmented BRAF V600E -induced migration/invasion in thyroid cells
To determine the cytotoxicity of BPA, Nthy-ori 3-1 cell line was treated with different concentrations of BPA ranging from 10 -10 to Compared with the control, Nthy-Vector and Nthy-BRAF V600E cells exposed to 10 -7 M BPA migrated to the scratch and healed the wound more quickly (Figure 4B), while cell migration rate was markedly declined after 3.33 × 10 -4 M BPA treatment ( Figure 4C). Accordingly, we concluded that 10 -7 M BPA could promote the migration of Nthy-  In order to explore the roles of ERK1/2-Cox2 signal in the EMT process induced by BPA exposure in Nthy-BRAF V600E cells, we subsequently detected the expression of several key molecules in this pathway and observed a significantly increased p-ERK and Cox2 levels ( Figure 5F-5H), Data presented as mean ± SD. *P < .05, **P < .01; # P < .05, ## P < .01 vs. the control group BPA in the above two cells( Figure 5F and 5G). Remarkably, we observed a combinative effect of BRAF V600E mutation and 10 -7 M BPA exposure on the activation of the ERK-Cox2 pathway ( Figure 5F-5H).

| D ISCUSS I ON
The BRAF V600E mutation as the most common genetic alteration in PTC and PTC-derived undifferentiated thyroid carcinoma contributes to a constitutively active onco-kinase 9,20 and is associated with mutation could make the thyroid cells acquire some typical phenotype of mesenchymal cell, such as more elongated shape and less intercellular contacts, as reported the alteration in cell morphology during the EMT process mainly induced by the conversion of cytoskeletal proteins from keratin to vimentin. 24 Notably, our in vitro data reminded 10 -7 M BPA exposure, a common human exposed dose could facilitate the process of EMT especially in PTC harbouring BRAF V600E mutation.
Overall less is known about the exact mechanisms by which BPA exposure could promote the EMT process of BRAF mutated thyroid cells. In light of previous research, BPA regulated Cox2-mediated EMT in hemangioma cells, 25 and BRAF V600E -induced EMT was dependent on the RAF-MEK-ERK signalling pathway, 26,27 our study introduced the ERK-Cox2 signal pathway to explain the potential mechanism. As expected, our data further clarified whether BPA exposure or BRAF V600E induced EMT was mediated by activating the ERK-Cox2 signalling pathway to enhance migration, invasion and clonality in thyroid cells, especially at 10 -7 M BPA exposure. However, 10 -4 M BPA treatment led to an inhibitory effect on the migration, invasion and clonality due to higher toxicity in thyroid cells. Undoubtedly, 10 -7 M BPA exposure will bring more serious health risks to PTC patients carrying BRAF V600E mutation. As 10 -7 M BPA exposure as a human exposed dose is of vital importance, our current study mainly focused on the health effects at such an exposed dose. Accordingly, it is necessary to suggest that patients suffering from thyroid disease should minimize the use of tableware and plastic products containing BPA in their daily lives. 28 Taken together, this study has focused on the interaction between BPA exposure and BRAF V600E mutation on the EMT progress of PTC for the first time. Remarkably, BPA exposure at a human exposed dose can synergize with the BRAF V600E mutation to promote EMT phenotype formation of thyroid cells by activating the ERK-Cox2 signalling pathway and further accelerate the migration/invasion of PTC. Although there are some limitations, our current study may contribute to understanding some shallow mechanisms underlying the combined effect of genetic mutation and endocrine-disrupting chemicals exposure.

ACK N OWLED G EM ENTS
This study was supported by grants from the National Natural

CO N FLI C T O F I NTE R E S T
No conflicts of interest exist regarding this study.