Abstract
We previously reported that ABI3 expression was decreased in thyroid cancer tissues and that ectopic expression of ABI3 in a follicular thyroid carcinoma cell line delayed cell cycle progression and inhibited cell proliferation, invasion, migration and tumor formation in athymic mice. These data indicated that ABI3 is a tumor suppressor gene; however the mechanism through which ABI3 is silenced in thyroid carcinomas is unknown. We here show that treatment of four follicular thyroid carcinoma cell lines with 5-aza-dC induced demethylation of a specific region of the ABI3 promoter and restored ABI3 expression. In contrast, 5-aza-dC treatment did not restore ABI3 expression in a non-thyroid cell line, suggesting a tissue-specific regulation. We additionally show that 8 CpG sites located within the ABI3 promoter are hypermethylated in most thyroid carcinoma samples and the degree of methylation correlated with ABI3 expression. Narrowing the region to specific CpG sites, the CpG4-6 sites showed the largest difference between benign and malignant lesions. In silico analysis revealed that these CpG sites flank a canonical binding site for NKX2-1, a thyroid specific transcriptional factor. Analysis of thyroid samples shows a correlation between NKX2-1 and ABI3 expression. In vitro assays demonstrate that NKX2-1 was required for ABI3 expression. Luciferase assay further confirmed the promoter activity of this region, which was increased when the cells were co-transfected with NKX2-1. Our study shows that the transcriptional silencing of ABI3 in cancer cells occurs via methylation and uncovered a previously unrecognized role for NKX2-1 in the regulation of ABI3.
Highlights
We previously reported that ABI3 expression is reduced or lost in follicular cell-derived thyroid carcinomas as compared to normal tissues and follicular thyroid adenomas (FTA) [1]
We show that 8 CpG sites located within the ABI3 promoter are hypermethylated in most thyroid carcinoma samples and the degree of methylation correlated with ABI3 expression
Restoration of ABI3 expression in thyroid carcinoma cells by 5-aza-dC treatment confirmed a causal correlation between DNA hypermethylation and ABI3 silencing in thyroid cell lines
Summary
We previously reported that ABI3 expression is reduced or lost in follicular cell-derived thyroid carcinomas as compared to normal tissues and follicular thyroid adenomas (FTA) [1]. We further demonstrated that ectopic expression of ABI3 inhibited cell proliferation, invasion, migration and delayed cell cycle progression of thyroid carcinoma cell line in vitro. ABI3 expression inhibited tumor formation in athymic mice [1]. These findings provide evidences that ABI3 is a tumor suppressor gene that plays important roles in the malignant transformation of thyroid tumors. Loss of ABI3 expression was reported in several cancer cell lines, including a highly metastatic U87 human glioma cell line. The authors further showed that forced expression of ABI3 into U87 cells suppressed cell motility and metastatic dissemination in vivo [2]
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