Multiple pluripotent stem cell markers in human anaplastic thyroid cancer: the putative upstream role of SOX2

Abstract

Background: Anaplastic thyroid carcinoma (ATC) is a rare and aggressive endocrine tumor with highly undifferentiated morphology. It has been suggested that cancer stem cells (CSCs) might play a central role in ATC. The objectives of this study were (i) to characterize CSCs from ex vivo ATC specimens by investigating the expression of several pluripotent stem cell markers, and (ii) to evaluate in vitro drug resistance modifications after specific CSC transcription factor switch-off.

Methods: In ex vivo experiments, eight formalin-fixed, paraffin-embedded ATC specimens were analyzed by reverse-transcription and real-time quantitative PCR and immunohistochemistry. In in vitro experiments using ATC SW1736 cells, the expression levels of OCT-4, NANOG, and ABCG2 and the sensitivity to either cisplatin or doxorubicin were evaluated after silencing.

Results: OCT-4, KLF4, and SOX2 transcription factors and C-KIT and THY-1 stem surface antigens showed variable up-regulation in all ATC cases. The SW1736 cell line was characterized by a high percentage of stem population (10.4±2.1% of cells were aldehyde dehydrogenase positive) and high expression of several CSC markers (SOX2, OCT4, NANOG, C-MYC, and SSEA4). SOX2 silencing down-regulated OCT-4, NANOG, and ABCG2. SOX2 silencing sensitized SW1736 cells, causing a significant cell death increase (1.8-fold) in comparison to control cells with 10 μM cisplatin (93.9±3.4% vs. 52.6±9.4%, p<0.01) and 2.7 fold with 0.5 μM doxorubicin (45.8±9.9% vs. 17.1±3.4% p<0.01). ABCG2 silencing caused increased cell death with both cisplatin (74.9±1.4%) and doxorubicin treatment (74.1±0.1%) vs. no-target-treated cells (respectively, 45.8±1.0% and 48.6±1.0%, p<0.001).

Conclusions: The characterization of CSCs in ATC through the analysis of multiple pluripotent stem cell markers might be useful in identifying cells with a stem-like phenotype capable of resisting conventional chemotherapy. In addition, our data demonstrate that SOX2 switch-off through ABCG2 transporter down-regulation has a major role in overcoming CSC chemotherapy resistance.

FIG. 1.

RT-PCR (A) and qRT-PCR (B, C) analysis for mature and fetal thyrocyte markers. Absence of TG, TPO, and NIS confirms the undifferentiated status of ATC specimens (A: lanes 1–8, ATC; lane 9, normal thyroid; 10, negative control). Expression of onfFN, PAX-8, and TTF-1 proves the thyroidal origin of the tumors (B, C). Values are shown as mean±SE. ATC, anaplastic thyroid carcinoma; bp, base pairs; RT-PCR, reverse-transcription PCR; qRT-PCR, real-time quantitative PCR; TG, thyroglobulin; TPO, thyroperoxidase; NIS, sodium/iodide symporter.

FIG. 2.

Expression of pluripotent stem cell nuclear transcription factors and surface stem cell markers in ATC. qRT-PCR analysis of nuclear stem cell transcription factors (A–D) and surface stem cell markers (E–F). Values were normalized to normal thyroid (see Methods) and shown as mean±SE. Data are representative of three independent experiments.

FIG. 3.

Co-localization of SOX2 and SSEA4. (A) Confocal analysis: surface stem cell marker SSEA4 (green) and nuclear stem cell marker SOX2 (red) co-localized. Counterstaining was performed with 4′,6-diamidino-2-phenylindole (DAPI, blue). For image acquisition and magnification see methods. (B) Immunohistochemistry: SSEA4 and SOX2 were strongly positive in the same four cases (20× magnification).

FIG. 4.

(A) SW1736 cell line characterization. RT-PCR analysis of thyrocyte markers in SW1736 cells. Lane 1, normal thyroid; lane 2, SW1736 cell line; lane 3, negative control; bp, base pairs. (B) Western blot analysis of AKT and p-AKT. (C) qRT-PCR analysis of relative expression of SOX2, OCT-4, NANOG, and C-MYC in the SW1736 cell line (□) vs. normal thyroid (■). Data are representative of three independent experiments. Values are shown as mean±SE. **p<0.001, ***p<0.0001. (D) FACS analysis of SW1736 cells using ALDEFLUOR assay. Thyroid cancer cells exposed to ALDEFLUOR substrate and a specific inhibitor of aldehyde dehydrogenase (ALDH) (i) were used to define the population with high ALDH activity (ii). SSC-H, side light scatter; FL1-H, fluorescence channel.

FIG. 5.

Analysis of SOX2 silencing. qRT-PCR analysis in the SW1736 cell line (A); SOX2 (B), NANOG (C), ABCG2 (D), and OCT-4 (E) expression in the SW1736 cell line after SOX2 silencing with stealth siRNA (siSOX2) vs. siCONTROL treated cells (Control). Western blot (F, G) of SOX2, OCT-4, and NANOG with proteins extracted from SW1736 cells after SOX2 silencing with stealth siRNA (□) vs. siCONTROL treated cells (■). Analysis of OCT4 silencing: OCT4 (H) and SOX2 (I) expression in the SW1736 cell line after OCT-4 silencing with siRNA (siOCT-4) vs. siCONTROL treated cells (Control). Data are representative of three independent experiments. Values are shown as mean±SE. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001. OD, optical density.

FIG. 6.

Effects of SOX2 and ABCG2 silencing on chemosensitivity. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis after cisplatin (i) and doxorubicin (ii) treatment of SW1736 cells after SOX2 (A) or ABCG2 (B) silencing with stealth siRNA (siRNA, ●) vs. siCONTROL treated cells (Control, ■). Data are representative of three independent experiments. Values are shown as mean±SE. *p<0.05, **p<0.01, ***p<0.001.