Int J Thyroidol.  2021 Nov;14(2):152-169. 10.11106/ijt.2021.14.2.152.

Cooperative Subtype Switch of Thyroid Hormone Receptor and Nuclear Receptor Corepressor Related Epithelial–Mesenchymal Transition in Papillary Thyroid Cancer

• Jeong S
• Lee SG
• Kim H
• Lee G
• Park S
• Kim IK
• Lee J
• Jo YS

• KMID: 2522920
• DOI: http://doi.org/10.11106/ijt.2021.14.2.152

Abstract

Background and Objectives
Although thyroid hormones affect human cancer progression, the regulatory mechanism of thyroid hormone receptors in carcinogenesis has not been elucidated. This study aimed to evaluate the expression pattern of the thyroid hormone receptor (TR) and its corepressors, and to investigate the clinical and biological functions of TR.
Materials and Methods
Transcriptomic and clinical data for thyroid cancer were downloaded from The Cancer Genome Atlas. Paraffin-embedded tissue sections from patients who underwent thyroidectomy were used for immunohistochemistry. BCPAP cells were treated with T3 to investigate the thyroid hormone target genes. Thyroid hormone receptor alpha (THRA) and Thyroid hormone receptor beta (THRB) were knocked down by transient siRNA transfection.
Results
THRA and THRB expression was lower in thyroid cancer tissues than in normal tissues. However, strong focal staining of TRβ was observed in the invasive front. High THRB expression was associated with high Silencing Mediator for Retinoid or Thyroid hormone receptor (SMRT) expression, older age, a high MACIS (distant Metastasis, patient Age, Completeness of resection, local Invasion, and tumor Size) score, more aggressive histological subtypes, more frequent extra-thyroidal extension, and advanced TNM stage. THRB expression was positively correlated with Hypoxia Inducible Factor 1 Subunit Alpha (HIF1A), L1 Cell Adhesion Molecule (L1CAM), and Lysyl Oxidase (LOX) expression. Thyroid hormone-induced HIF1A, L1CAM, and LOX upregulation was abolished by siTHRB but not siTHRA in BCPAP cells. High SMRT and high THRB groups (SMRT/THRB) presented more aggressive clinical features and showed an upregulation of HIF1A, L1CAM, and LOX, as well as of epithelial-mesenchymal transition (EMT)-related genes, causing changes in the tumor microenvironment.
Conclusion
Cooperative subtype switching from NCOR1/THRA to SMRT/THRB was thus related to aggressive clinical and molecular features, possibly related to EMT and EMT-related tumor microenvironment.

Keyword

Thyroid hormone receptors; Nuclear receptor co-repressors; Epithelial-mesenchymal transition; Tumor microenvironment

Figure

• 

  Fig. 1 Comparison of (A) THRA, (B) THRB, (C) NCOR1 and (D) SMRT mRNA expression between normal and thyroid cancer tissues by unpaired (left panel) and paired (right panel) t tests. NCOR1: nuclear receptor corepressor 1, SMRT: silencing mediator for retinoid or thyroid hormone receptor, THRA: thyroid hormone receptor alpha, THRB: thyroid hormone receptor beta. Data are presented as mean± S.D. Mean comparisons were analyzed by an unpaired or paired t test. *p<0.0001.

• 

  Fig. 2 Representative immunohistochemistry-paraffin (IHC-P) staining for TRα (A), TRβ (B) in papillary thyroid cancer. THRA: thyroid hormone receptor alpha, THRB: thyroid hormone receptor beta. Red boxes indicate the areas magnified at the next high power field. Scale bars indicate 25 μm. Red arrows indicate the focal strong staining intensity of TRβ in the invasive front area.

• 

  Fig. 3 TRβ dependent upregulation of HIF1A target genes in PTC. (A) Correlation between expression levels of THRA, THRB and HIF1A in TCGA (The Cancer Genome Atlas) THCA (thyroid carcinoma). (B) Representative results of reverse transcription-polymerase chain reaction (RT-PCR) indicating the effect of T3 on HIF1A target genes. (C) Representative results of RT-PCR showing the silencing efficacy effect of siTHRA and siTHRB. (D) Representative results of RT-PCR showing the effect of THRA, THRB silencing on the expression of HIF1A target genes in BCPAP cells. HIF1A: hypoxia inducible factor1 alpha, T3: triiodothyronine, THRA: thyroid hormone receptor alpha, THRB: thyroid hormone receptor beta. All experiments were repeated three times, and each experiment was performed in triplicate. Correlation coefficients were calculated by Pearson’s method.

• 

  Fig. 4 Positive relationship of THRB expression with EMT-related genes. (A) Correlation between expression levels of THRA, THRB and epithelial to mesenchymal transition genes in TCGA THCA. (B) qPCR analysis presenting T3 induced upregulation of EMT-related genes in BCPAP cells. (C) Comparison of the expression of HIF1A target genes according to subtype switch from high NCOR1/high THRA (NCOR1-THRA) to high SMRT/high THRB (SMRT/THRB). Comparison of (D) immune score, (E) stromal score, and (F) ESTIMATE score according to subtype switch. EMT: epithelial- mesenchymal transition, HIF1A: hypoxia inducible factor1 alpha, NCOR1: nuclear receptor corepressor 1, SMRT: silencing mediator for retinoid or thyroid hormone receptor, TCGA: The Cancer Genome Atlas, THCA: thyroid carcinoma, THRA: thyroid hormone receptor alpha, THRB: thyroid hormone receptor beta. All experiments were repeated three times, and each experiment was performed in triplicate. Data are presented as mean±S.D. Correlation coefficients were calculated by Pearson’s method. Mean comparisons were analyzed by an unpaired or paired t test. *p<0.01; **p<0.001; ***p< 0.0001.

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