Cancer Res Treat.  2017 Apr;49(2):509-517. 10.4143/crt.2016.214.

The Role of Notch1 Signaling in Anaplastic Thyroid Carcinoma

• Kim HJ
• Kim MJ
• Kim A
• Jung CW
• Park S
• Koh JS
• Myung JK

• KMID: 2378124
• DOI: http://doi.org/10.4143/crt.2016.214

Abstract

PURPOSE
The Notch signaling pathway is widely expressed in normal, reactive, and neoplastic tissues; however, its role in thyroid tissues has not been fully elucidated. Therefore, this study was conducted to characterize the expression of the Notch signaling pathway in papillary thyroid cancer (PTC) cells and anaplastic thyroid cancer (ATC) cells.
MATERIALS AND METHODS
Expression of activated Notch1 in ATC and PTC paraffin-embedded tissues was determined by immunohistochemistry. The small interfering RNA techniquewas employed to knock down Notch1 expression in ATC and PTC cell lines.
RESULTS
The expression of activated Notch1 was higher in ATC cases than in PTC cases. Inhibition of Notch1 significantly reduced proliferation and migration of ATC cells, but not PTC cells. In addition, inhibition of Notch1 in ATC cells significantly reduced the expression of key markers of epithelial-mesenchymal transition and cancer stem cells. Conversely, changes in the expression of these proteins were not observed in PTC cells.
CONCLUSION
The results of this study suggest that Notch1 expression plays different roles in tumor progression in ATC and PTC cells. We also found that Notch1 expression was significantly related to the highly invasive or proliferative activity of ATC cells.

Keyword

Receptor Notch1; Anaplastic thyroid carcinoma; Papillary thyroid cancer; Epithelial-mesenchymal transition; Neoplastic stem cells

MeSH Terms

Cell Line
Epithelial-Mesenchymal Transition
Immunohistochemistry
Neoplastic Stem Cells
RNA, Small Interfering
Thyroid Carcinoma, Anaplastic*
Thyroid Gland
Thyroid Neoplasms
RNA, Small Interfering

Figure

• 

  Fig. 1. Expression of activated Notch1 in patient tissues and thyroid cancer cell lines. (A) Immunohistochemical analysis of activated Notch1 expression in anaplastic (upper left, negative; upper right, positive; ×400) and papillary (lower left, negative; lower right, positive; ×400) thyroid cancer tissues. (B) Western blot analysis of activated Notch1, Notch1, and Notch2 expression in BCPAP and 8505C thyroid cancer cell lines. Each graph shows the target protein expression ratio normalized to β-actin. Statistical significance is based on the difference when compared with BCPAP cells, *p < 0.05. ATC, anaplastic thyroid cancer; PTC, papillary thyroid cancer.

• 

  Fig. 2. Knockdown of Notch1 reduced cell viability in anaplastic thyroid cancer cell lines. 8505C and BCPAP cells were transfected with si-control or si-Notch1 for 72 hours, after which proliferation was determined by cell counting (A) and propidium iodide uptake (B). Statistical significance is based on the difference when compared with si-control cells, **p < 0.01, ***p < 0.001.

• 

  Fig. 3. Knockdown of Notch1 inhibited cell migration in anaplastic thyroid cancer cell lines. For the analysis of cell migration, 8505C (A) and BCPAP (B) cells in which Notch1 was knocked down were plated into transwell inserts, and 10% fetal bovine serum was used in the medium in the bottom chambers. After 48 hours, migration of thyroid cancer cells was quantified (×100). Statistical significance is based on the difference when compared with si-control cells, **p < 0.01, ***p < 0.001.

• 

  Fig. 4. DAPT reduced cell viability and cell migration in anaplastic thyroid cancer cell lines. (A) 8505C and BCPAP cells were treated with various concentrations of DAPT for 72 hours, after which proliferation was determined by cell counting. (B) The migration cells were stained with crystal violet and the images were photographed (×100). Statistical significance is based on the difference when compared with untreated cells, **p < 0.01, ***p < 0.001. DAPT, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester.

• 

  Fig. 5. Knockdown attenuated cancer stem cell (CSC)– and epithelial-mesenchymal transition (EMT)–related markers. 8505C and BCPAP cells were transfected with si-control or si-Notch1 for 72 hours. Total cell lysates were prepared, Notch1, Notch2, activated Notch1, activated Notch2 (A), and CSC- and EMT-related protein expression (B) were detected by western blot analysis.

Reference

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