Bisphenol A at a human exposed level can promote epithelial-mesenchymal transition in papillary thyroid carcinoma harbouring BRAFV600E 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 BRAF^V600E 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.

Keywords: BRAFV600E mutation; Bisphenol A; ERK-Cox2; Papillary thyroid carcinoma; epithelial-mesenchymal transition.

FIGURE 1

BRAF ^V600E mutation‐induced EMT in PTC patients. (A, D and G) Typical images of PTC tissue with strong positive, positive, weak positive and negative staining of E‐cadherin (A), N‐cadherin (D) and MMP‐9 (G) in proper order. (B, E and H) Representative for the protein expression of E‐cadherin, N‐cadherin and MMP‐9, respectively, in the carcinoma tissues of PTC patients with wide‐type or mutated BRAF ^V600E. (C, F and I) Representative for the mRNA expression of E‐cadherin, N‐cadherin and MMP‐9, respectively, in the carcinoma tissues of PTC patients with wide‐type or mutated BRAF ^V600E. *P < .05, **P < .01

FIGURE 2

BPA exposure enhanced EMT in PTC patients harbouring BRAF ^V600E mutation. (A‐C) Representative for the IHC‐P Score of E‐cadherin, N‐cadherin and MMP‐9. (D‐F) Representative for the mRNA expression of E‐cadherin, N‐cadherin and MMP‐9. *P < .05

FIGURE 3

Establishment of the BRAF ^V600E overexpressed cell model. (A) Typical fluorescence images of Nthy‐ori 3‐1 cells after 48h's transfection with different plasmids. (B) The mRNA level of BRAF gene after 48h's transfection in Nthy‐ori 3‐1 cells. (C) Protein expression levels of BRAF, p‐ERK, ERK after 48h's transfection in Nthy‐ori 3‐1 cells, normalized to GAPDH. (D‐F) Typical images of parental Nthy‐ori 3‐1 cells (D) and cells transfected with control (E) or BRAF ^V600E mutated plasmid (F). The yellow arrow in the figure (F) marked the spindle transformed shape cells. **P < .01

FIGURE 4

BPA exposure augmented BRAF ^V600E‐induced migration/invasion of Nthy‐ori 3‐1 cells. (A‐C) Typical images of the scratch assay in Nthy‐ori 3‐1 cells transfected with different plasmids combined exposure to BPA at the concentrations of 0, 10^‐7 M and 3.33 × 10^‐4 M, respectively. Scale bars are 400 µm. (D) Representative for the migration rate of scratch assay in transfected cells treated with different concentrations of BPA. (E) Typical images of Transwell migration experiment in transfected cells treated with different concentrations of BPA. Scale bars are 400µm. (F) Representative for the number of migrated cells in transfected cells treated with different concentrations of BPA. (G) Typical images of Transwell invasion assay in transfected cells treated with different concentrations of BPA. (H) Representative for the number of invading cells in transfected cells treated with different concentrations of BPA. (I) Typical images of colony‐forming assay in transfected cells treated with different concentrations of BPA. (J) Qualification of the above colony‐forming assay. Data presented as mean ± SD. *P < .05, **P < .01, ^# P < .05 vs. the control group

FIGURE 5

BPA exposure accelerated BRAF ^V600E‐induced EMT of Nthy‐ori 3‐1 cell via activating the ERK‐Cox2 signalling pathway. (A) The protein expressions of BRAF, MMP‐9, E‐cadherin and N‐cadherin proteins in transfected cells treated with different concentrations of BPA. (B‐E) Relative intensity of BRAF, E‐cadherin, N‐cadherin and MMP‐9 protein expression. (F) The protein expression of BRAF p‐ERK, ERK and Cox2 in transfected cells treated with different concentrations of BPA. (G‐H) Relative intensity of p‐ERK/ERK and Cox2 protein expression. Data presented as mean ± SD. *P < .05, **P < .01; ^# P < .05, ^## P < .01 vs. the control group