Targeting GLI1 Transcription Factor for Restoring Iodine Avidity with Redifferentiation in Radioactive-Iodine Refractory Thyroid Cancers

Abstract

Radioactive-iodine (RAI) therapy is the mainstay for patients with recurrent and metastatic thyroid cancer. However, many patients exhibit dedifferentiation characteristics along with lack of sodium iodide symporter (NIS) functionality, low expression of thyroid-specific proteins, and poor RAI uptake, leading to poor prognosis. Previous studies have demonstrated the effect of GLI family zinc finger 1 (GLI1) inhibition on tumor growth and apoptosis. In this study, we investigated the role of GLI1 in the context of redifferentiation and improvement in the efficacy of RAI therapy for thyroid cancer. We evaluated GLI1 expression in several thyroid cancer cell lines and selected TPC-1 and SW1736 cell lines showing the high expression of GLI. We performed GLI1 knockdown and evaluated the changes of thyroid-specific proteins expression, RAI uptake and I-131-mediated cytotoxicity. The effect of GANT61 (GLI1 inhibitor) on endogenous NIS expression was also assessed. Endogenous NIS expression upregulated by inhibiting GLI1, in addition, increased expression level in plasma membrane. Also, GLI1 knockdown increased expression of thyroid-specific proteins. Restoration of thyroid-specific proteins increased RAI uptake and I-131-mediated cytotoxic effect. Treatment with GANT61 also increased expression of endogenous NIS. Targeting GLI1 can be a potential strategy with redifferentiation for restoring RAI avidity in dedifferentiated thyroid cancers.

Keywords: GLI1; radioactive-iodine therapy; redifferentiation; sodium iodide symporter; thyroid cancer.

Figure 1

Investigation of hedgehog signaling pathway in thyroid cancer-derived cell lines. Three papillary thyroid cancer cell lines (BCPAP, K1, TPC-1) and three anaplastic thyroid cancer cell lines (BHT101, CAL62, SW1736) were subjected to western blot analysis. (A) Evaluation of the hedgehog signaling pathway. (B) Quantitative analysis of GLI1 expression in several thyroid cancer cell lines. ß-actin was used as an internal control. Mean ± standard deviation (SD) values from three independent experiments are presented. (C) Confirmation of NIS protein expression level in thyroid cancer-derived cell lines. ß-actin was used as an internal control. Raw data is presented in Figures S2–S4.

Figure 2

Changes in endogenous NIS expression and its localization in GLI1-inhibited thyroid cancer cells. Both TPC-1 and SW1736 cells were treated with scrambled siRNA, GLI1 siRNA or NIS siRNA for 48 h. (A) Changes in NIS and GLI1 expression induced by GLI1 siRNA treatment in TPC-1 cells. GAPDH was used as an internal control. Mean ± SD values from five independent experiments are presented. *** p < 0.001 (Student’s t-test). (B) Evaluation of NIS expression via GLI1 knockdown in SW1736 cells. Mean ± SD values from five independent experiments are presented. * p < 0.05 (Student’s t-test). Western blot analysis for NIS protein with scrambled siRNA, GLI1 siRNA or NIS siRNA treatment to TPC-1 cells (C) and SW1736 cells (D). Immunofluorescence images showing localization of endogenous NIS expression in TPC-1 cells (E) and SW1736 cells (F). Scale bar: 20 µm. Expression of endogenous NIS protein in plasma membrane fraction by GLI1 knockdown in TPC-1 cells (G) and SW1736 cells (H). Caveolin-1 were used as loading controls for plasma membrane proteins. Raw data is presented in Figures S5–S10.

Figure 3

Evaluation of the expression of thyroid-specific proteins and transcription factors in thyroid cancer-derived cells with GLI1 knockdown. Both TPC-1 and SW1736 cells were treated with scrambled siRNA or GLI1 siRNA for 48 h and changes in the expression of thyroid-specific proteins were evaluated. (A) Western blot analysis showing expression of thyroid-specific proteins (thyroperoxidase (TPO) and TSH receptor (TSHR)) and transcription factors (PAX-8 and TTF-1) in TPC-1 cells. (B) Quantitative analysis of western blots. GAPDH was used as an internal control. Mean ± SD values from at least three independent experiments are reported. * p < 0.05, ** p < 0.01 (Student’s t-test). (C) Changes of thyroid-specific proteins and transcription factors expression in SW1736 cells with GLI1 knockdown. (D) Quantitative analysis of thyroid-specific proteins and transcription factors expression in SW1736 cells. Mean ± SD values from at least three independent experiments are presented. * p < 0.05, *** p < 0.001 (Student’s t-test). Raw data is presented in Figures S11–S14.

Figure 4

Verification of the extent of I-125 accumulation and I-131-mediated cytotoxic effect in GLI1-inhibited thyroid cancer cells. Both TPC-1 and SW1736 cells were treated with scrambled siRNA or GLI1 siRNA for 48 h. (A) For I-125 uptake assay, cells were treated with 37 kBq carrier-free I-125 and 100 μM sodium iodide at 37 °C for 30 min. Potassium perchlorate (KCIO₄) was used as a competitive inhibitor of iodide transport. Upper–TPC-1 cells; Lower–SW1736 cells. The results are expressed as mean ± SD values of the experiment performed in triplicates. * p < 0.05, NS: Not Significant (by Student’s t-test). (B) After incubation of siRNA in TPC-1 cells, the cells were incubated with or without 50 µCi/ml I-131 supplemented with 30 μM NaI for 7 h at 37 °C. Images about I-131 clonogenic assay. (C) Quantitative analysis based on I-131 clonogenic assay. The results are expressed as mean ± SD values of the experiment performed in triplicates. *** p < 0.001, ** p < 0.01, NS: Not Significant (by Student’s t-test).

Figure 5

Efficacy of GANT61 in restoring endogenous NIS expression in thyroid cancer cells. Both TPC-1 and SW1736 cells were exposed to GANT61. (A) Results of cell viability assay showing time- and dose-dependent effects of GANT61 in TPC-1 cells. Mean ± SD values from three optical density (OD) is reported. (B) Endogenous NIS expression in whole cell lysate after treatment with GANT61. GAPDH was used as a loading control. The results are expressed as mean ± SD values of the experiment performed in quintuplicates. * p < 0.05, ** p < 0.01, NS: Not Significant (by Student’s t-test). (C) Immunofluorescence images for monitoring changes of expression and localization with endogenous NIS in thyroid cancer-derived cells treated with GANT61. (D) Change of endogenous NIS expression in plasma membrane proteins. Caveolin-1 were used as loading controls for plasma membrane proteins. Raw data is presented in Figures S15 and S16.