Endocrinol Metab.  2022 Dec;37(6):879-890. 10.3803/EnM.2022.1563.

BRAF^V600E Mutation Enhances Estrogen-Induced Metastatic Potential of Thyroid Cancer by Regulating the Expression of Estrogen Receptors

• Kim M
• Kim Sj
• Ha SY
• Xu Z
• Han Y
• Jee HG
• Cho SW
• Park YJ
• Lee KE

• KMID: 2537289
• DOI: http://doi.org/10.3803/EnM.2022.1563

Abstract

Background
Cross-talk between mitogen-activated protein kinase and estrogen has been reported; however, the role of BRAF^V600E in the estrogen responsiveness of thyroid cancer is unknown. We elucidated the effect of BRAF^V600E on the estrogen-induced increase in metastatic potential in thyroid cancer.
Methods
Using a pair of cell lines, human thyroid cell lines which harbor wild type BRAF gene (Nthy/WT) and Nthy/BRAF^V600E (Nthy/V600E), the expression of estrogen receptors (ERs) and estrogen-induced metastatic phenotypes were evaluated. Susceptibility to ERα- and ERβ-selective agents was evaluated to confirm differential ER expression. ESR expression was analyzed according to BRAF^V600E status and age (≤50 years vs. >50 years) using The Cancer Genome Atlas (TCGA) data.
Results
Estradiol increased the ERα/ERβ expression ratio in Nthy/V600E, whereas the decreased ERα/ERβ expression ratio was found in Nthy/WT. BRAF^V600E-mutated cell lines showed a higher E2-induced increase in metastatic potential, including migration, invasion, and anchorage-independent growth compared with Nthy/WT. An ERα antagonist significantly inhibited migration in Nthy/V600E cells, whereas an ERβ agonist was more effective in Nthy/WT. In the BRAF^V600E group, ESR1/ESR2 ratio was significantly higher in younger age group (≤50 years) compared with older age group (>50 years) by TCGA data analysis.
Conclusion
Our data show that BRAF^V600E mutation plays a crucial role in the estrogen responsiveness of thyroid cancer by regulating ER expression. Therefore, BRAF^V600E might be used as a biomarker when deciding future hormone therapies based on estrogen signaling in thyroid cancer patients.

Keyword

Thyroid neoplasms; Proto-oncogene proteins B-raf; Estrogens; Receptors, estrogen; Neoplasms

Figure

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  Fig. 1. Expression change of estrogen receptor α (ERα) and ERβ in thyroid cell lines by E2 treatment depending on BRAFV600E status. (A) Expression of estrogen receptors were measured to see how BRAFV600E affects regulation of the receptors in thyroid cell lines. All cell lines were cultured in phenol red-free complete medium and treated with 100 nM of E2 for 24 hours. The response to E2 treatment was opposite between human thyroid cell lines which harbor wild type BRAF gene (Nthy/WT) and Nthy/BRAFV600E (Nthy/V600E). It shows increased ERα and decreased ERβ in Nthy/V600E whereas decreased ERα and increased ERβ in Nthy/WT by E2 treatment. (B) Densitometry results for fold change of ERα, ERβ, and the ratio of ERα over ERβ after E2 treatment. Each value was calculated by fold change of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-normalized levels of ER proteins on the same membranes. E2 treatment induced decreased ER ratio in Nthy/WT, but increased in Nthy/V600E (0.50 vs. 1.67, P=0.0006). aP<0.01; bP<0.001.

• 

  Fig. 2. Enhanced increase of metastatic potentials of thyroid cell lines by BRAFV600E. (A) Human thyroid cell lines which harbor wild type BRAF gene (Nthy/WT) did not show significant changes of migration and invasion by E2. However, BRAFV600E-positive cell lines, and Nthy/BRAFV600E (Nthy/V600E), showed over two times more increase of migration and significant increase of invasion in response to E2 treatment. (B) Anchorage-independent growth of thyroid cell lines was observed under microscopy and the total amount of cells was measured using fluorescent dye included in the soft-agar assay kit. While E2 slightly inhibited growth of Nthy/WT, it promoted colony formation and growth in BRAFV600E-positive cell lines in a dose-dependent manner. NS, not significant. aP<0.05.

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  Fig. 3. Altered anti-migratory effects of selective estrogen receptor α (ERα) antagonist and ERβ agonist. Wound healing assay was used to see if susceptibilities to diarylpropionitrile (DPN) and methylpiperidino pyrazole (MPP), which activate ERβ and block ERα, respectively. Both result in inhibition of cell migration. Under presence of endogenous estrogen and estrogenic phenol red, treatment of the reagents showed different efficacies depending on BRAFV600E status. As the ratio of ERα over ERβ decreased in human thyroid cell lines which harbor wild type BRAF gene (Nthy/WT) under presence of E2, DPN, ERβ-selective agonist was more effective that MPP in Nthy/WT while Nthy/BRAFV600E (Nthy/V600E) showed the opposite. NS, not significant. aP<0.05; bP<0.01.

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  Fig. 4. Relative estrogen receptor 1 (ESR1) and ESR2 expression depending on BRAFV600E status and menopause-related age group. (A) Papillary thyroid cancer (PTC) patients (363 females) were divided according to BRAFV600E status, and we compared expression of ESR genes between age groups because the level of estrogen is known to dramatically decrease around age 50. Whereas BRAFWT group does no show significant differences in both ESR1 and ESR2 genes between age groups, BRAFV600E group shows significantly higher ESR1 and ESR1/2 ratio in younger group (age under 51). (B) Correlation between ESR genes and BRAFV600E-RAS score (BRS) in each group by BRAFV600E and age. In the BRAFWT group, there are no clear differences between the age groups, but the BRAFV600E group shows different associations depending on age group. BRAFV600E-like gene signature (higher BRS) is correlated positively with ESR1 and ESR1/2 ratio and negatively with ESR2 expression in premenopausal age group (age <51, purple line) whereas it was opposite in post-menopausal age group with BRAFV600E (age ≥51, blue line). (C) Heatmap to show ESR genes and other prognosis-related gene signatures according to the level of BRAFV600E-RAS signature in thyroid cancer. TCGA, The Cancer Genome Atlas; RSEM, RNA-sequencing by expectation maximization; NS, not significant. aP<0.05; bP<0.01.

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