Endocrinol Metab.  2019 Jun;34(2):203-212. 10.3803/EnM.2019.34.2.203.

Expression of NF2 Modulates the Progression of BRAFV600E Mutated Thyroid Cancer Cells

• You MH
• Jeon MJ
• Kim TY
• Kim WB
• Shong YK
• Kim WG

• KMID: 2450521
• DOI: http://doi.org/10.3803/EnM.2019.34.2.203

Abstract

BACKGROUND
We previously reported the frequent neurofibromatosis 2 (NF2) gene mutations in anaplastic thyroid cancers in association with the BRAF V600E mutation. We aimed to investigate the role of NF2 in thyroid cancer with BRAF mutation.
METHODS
To identify the function of NF2 in thyroid cancers, we investigated the changes in cell proliferation, colon formation, migration and invasion of thyroid cancer cells (8505C, BHT101, and KTC-1) with BRAF V600E mutation after overexpression and knock-down of NF2. We also examined how cell proliferation changed when NF2 was mutagenized. Human NF2 expression in papillary thyroid carcinoma (PTC) was analyzed using the The Cancer Genome Atlas (TCGA) data.
RESULTS
First, NF2 was overexpressed in 8505C and KTC-1 cells. Compared to control, NF2 overexpressed group of both thyroid cancer cells showed significant inhibition in cell proliferation and colony formation. These results were also confirmed by cell migration and invasion assay. After knock-down of NF2 in 8505C cells, there were no significant changes in cell proliferation and colony formation, compared with the control group. However, after mutagenized S288* and Q470* sites of NF2 gene, the cell proliferation increased compared to NF2 overexpression group. In the analysis of TCGA data, the mRNA expression of NF2 was significantly decreased in PTCs with lateral cervical lymph node (LN) metastasis compared with PTCs without LN metastasis.
CONCLUSION
Our study suggests that NF2 might play a role as a tumor suppressor in thyroid cancer with BRAF mutation. More studies are needed to elucidate the mechanism how NF2 acts in thyroid cancer with BRAF mutation.

Keyword

Neurofibromatosis 2; BRAF, mutation; Thyroid neoplasms; Genes, tumor suppressor

MeSH Terms

Cell Movement
Cell Proliferation
Colon
Genes, Neurofibromatosis 2
Genes, Tumor Suppressor
Genome
Humans
Lymph Nodes
Neoplasm Metastasis
Neurofibromatosis 2
RNA, Messenger
Thyroid Carcinoma, Anaplastic
Thyroid Gland*
Thyroid Neoplasms*
RNA, Messenger

Figure

• 

  Fig. 1 Neurofibromatosis 2 (NF2) overexpression suppresses cell growth in 8505C and KTC-1 cells. (A) Western blot analysis confirmed the differences in the Merlin protein expression between groups. (B) Proliferation assay to measure the cell growth rate in control and NF2 overexpressing 8505C cells. (C) Colony formation assay of 8505C cells in Day 7. (D) Proliferation assay to measure the cell growth rate in control and NF2 overexpressing KTC-1 cell. (E) Colony formation assay of KTC-1 cells in Day 7. Each data point represents mean±standard error of three independent experiments. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Vec, vector. aP<0.05.

• 

  Fig. 2 Neurofibromatosis 2 (NF2) overexpression inhibits cancer cell migration and invasion cancer in 8505C and KTC-1 cell. (A, B) In vitro wound healing assay in control and NF2 overexpressing thyroid cancer cells. Image was measured after 0, 24 hours after scratching (left) and quantification of wound closure was expressed in graph (right) (A) 8505C, (B) KTC-1. (C, D) In vitro transwells assay in control and NF2 overexpressing thyroid cancer cells. Representative images of invasive potential are shown and quantification was expressed in the bar graph (right) (C) 8505C, (D) KTC-1. The photographs were taken at 100×X and the bar size was 50 µm. Each data point represents mean±standard error of three independent experiments. Vec, vector. aP<0.05.

• 

  Fig. 3 Neurofibromatosis 2 (NF2) loss fails to suppress growth in KTC-1 cells. (A) Proliferation assay to measure the cell growth rate in the control and small interfering NF2 (siNF2) KTC-1 cells. (B) Colony formation assay in the control and siNF2 KTC-1 cells. After the NF2/control clustered regularly interspaced short palindromic repeats (CRISPR) were conducted in the KTC-1 cell line, the NF2 gene knock out rate were measured using (C) confocal image, (D) fluorescence activated cell sorter sorting, (E) proliferation assay to measure the cell growth rate in the control KTC-1 cells and NF2 knock down KTC-1 cells using CRISPR/Cas9 system. (F) Colony formation assay in KTC-1 cells using CRISPR/Cas9 system. Each data point represents mean±standard error of three independent experiments. NS, no significant; FSC-A, forward Scatter-A; FITC-A, fluorescin isothiocyanate-A.

• 

  Fig. 4 Neurofibromatosis 2 (NF2) mutagenesis enhances cell growth in 8505C and BHT101 cells. (A, B) Mutagenesis of the NF2 gene was performed on 8505C and BHT101. Cell proliferation was assessed in control and NF2 overexpressing thyroid cancer cells and also mutagenized thyroid cancer cells at 288 and 470 sites of NF2 gene. (A) 8505C: the control, 288, and 470 site of NF2 gene group showed significant decreased cell proliferation compared to NF2 group. (B) BHT101: the control, 288, and 470 site of NF2 gene group showed significant decreased cell proliferation compared to NF2 group. Each data point represents mean±standard error of three independent experiments. OD, optical density; pCMV, cytomegalovirus immediate early promoter. aP<0.001; bP<0.05.

• 

  Fig. 5 Association between neurofibromatosis 2 (NF2) expression and cervical lymph node (LN) metastasis of papillary thyroid cancer. By The Cancer Genome Atlas data, mRNA expression of NF2 was significantly different according to cervical LN metastasis status. Especially, mRNA expression of NF2 was significantly decreased in papillary thyroid carcinomas (PTCs) with lateral cervical LN metastasis, compared to PTCs without LN metastasis in both (A) BRAF wild-type group and (B) BRAFV600E mutant group. Y-axis showed log2 transformed mRNA expression of NF2. In post hoc analysis, mRNA expression of NF2 was significantly different between N0 and N1b group. aP<0.05.

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