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Published on: March 27, 2020
Transcriptional activation of the thyroglobulin promoter directing suicide gene expression by thyroid transcription
H Shimura1, H Suzuki, A Miyazaki
1Third Department of Internal Medicine, Yamanashi Medical University, Yamanashi 409-3898, Japan.
Abstract:
Gene therapy with thyroglobulin (TG) promoter and a prodrug/suicide gene combination may prove useful as a treatment for thyroid carcinoma. However, most poorly differentiated and anaplastic thyroid carcinomas have lost the ability to express the TG gene expression accompanied by loss of transcription factors [thyroid transcription factor-1 (TTF-1), TTF-2, or Pax-8] interacting with the TG promoter. In anticipation of developing transcriptionally targeted gene therapy of TG-nonproducing thyroid carcinomas, we investigated the effect of TTF-1 gene transfer on TG promoter activity and the cytotoxic effect obtained by the TG promoter-driven HSV-TK gene along with ganciclovir in thyroid carcinoma and nonthyroidal cells. Using a chimeric construct containing the 5'-flanking region of the rat TG gene between -826 and +39 bp and the luciferase gene, TG promoter activity was detected in a normal rat thyroid cell line (FRTL-5), but not in a dedifferentiated line of thyroid cells (FRT) expressing Pax-8 but not TTF-1, TTF-2, or TG [TTF-1(-)/TTF-2(-)/Pax-8(+)/TG(-)], or in a human papillary thyroid carcinoma cell line [BHP15-3; TTF-1(-)/TTF-2(-)/Pax-8(-)/TG(-)], a human pulmonary cell line [H441; TTF-1(+)/TTF-2(-)/Pax-8(-)/TG(-)], or a dog kidney epithelial cell line [MDCK; TTF-1(-)/TTF-2(-)/Pax-8(+)/TG(-)]. Cotransfection of the TTF-1 expression vector stimulated TG promoter activity in FRT and BHP15-3 dedifferentiated thyroid cells, but not in H441 pulmonary cells. Only weak activation was observed in MDCK kidney cells. We then constructed recombinant adenovirus vectors, AdTTF-1 and ADTGTK: AdTTF-1 contained cytomegalovirus promoter and rat TTF-1 cDNA; AdTGTK carried the TG promoter-driven HSV-TK gene. Infection with AdTGTK and combined with GCV treatment induced a cytotoxic effect in FRTL-5 cells but not in dedifferentiated thyroid or nonthyroid cells. Cotransduction of AdTTF-1 and AdTGTK permitted 90% cytotoxicity for BHP15-3 and >95% cytotoxicity for FRT, as well as for BHP7-13 and BHP18-21v thyroid cancer cell lines [both/TTF1(-)/TTF-2(-)/Pax-8(+)/TG(-)]. In contrast, little cytotoxicity was seen for H441 and MDCK cell lines even with 300 microg/ml of ganciclovir. These results suggest that cotransduction of a TG promoter-controlled suicide gene and the TTF-1 gene by adenoviral vectors confers transcriptionally targeted gene-mediated cytotoxicity in poorly differentiated thyroid carcinoma cells unable to express the TG gene.
Insights
Gene therapy using TTF-1 gene transfer and a suicide gene effectively targets thyroid cancer cells that don't produce thyroglobulin. This approach shows significant cytotoxicity in poorly differentiated thyroid carcinomas, offering a new treatment strategy.
Area of Science:
- Molecular Biology
- Oncology
- Gene Therapy
Background:
- Thyroid carcinoma treatment faces challenges with poorly differentiated and anaplastic types that lose thyroglobulin (TG) gene expression.
- Loss of TG expression is linked to reduced activity of key transcription factors like thyroid transcription factor-1 (TTF-1), TTF-2, and Pax-8.
- Targeting TG-nonproducing thyroid carcinomas requires strategies that overcome the lack of endogenous TG promoter activity.
Purpose of the Study:
- To investigate the effect of TTF-1 gene transfer on TG promoter activity in thyroid cancer cells.
- To evaluate the cytotoxic efficacy of a TG promoter-driven suicide gene (HSV-TK) combined with ganciclovir (GCV) in various cell lines.
- To develop a transcriptionally targeted gene therapy for TG-nonproducing thyroid carcinomas.
Main Methods:
- Utilized a luciferase reporter assay with a rat TG gene 5'-flanking region construct to assess promoter activity.
- Employed recombinant adenovirus vectors: AdTTF-1 (for TTF-1 expression) and AdTGTK (for TG promoter-driven HSV-TK).
- Assessed cytotoxicity through combined AdTTF-1 and AdTGTK cotransduction followed by ganciclovir treatment in differentiated, dedifferentiated, and nonthyroidal cell lines.
Main Results:
- TTF-1 gene transfer significantly stimulated TG promoter activity in dedifferentiated thyroid cells (FRT, BHP15-3) lacking endogenous TTF-1.
- Adenovirus-mediated TG promoter-driven HSV-TK and ganciclovir treatment showed cytotoxicity in normal thyroid cells (FRTL-5) but not in dedifferentiated or nonthyroidal cells.
- Cotransduction of AdTTF-1 and AdTGTK resulted in high cytotoxicity (>90%) in multiple TG-nonproducing thyroid cancer cell lines, with minimal effect on non-target cells.
Conclusions:
- Restoring TTF-1 expression can reactivate the TG promoter in dedifferentiated thyroid cancer cells.
- Adenoviral cotransduction of TTF-1 and a TG promoter-controlled suicide gene provides a transcriptionally targeted approach for gene therapy.
- This strategy demonstrates significant potential for selectively eliminating poorly differentiated thyroid carcinoma cells that lack TG gene expression.
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