Hypermethylation of a New Distal Sodium/Iodide Symporter (NIS) enhancer (NDE) is associated with reduced NIS expression in thyroid tumors.

CONTEXT

In thyroid tumors, reduced radioiodine uptake is associated with worse patient outcome concomitantly with low sodium/iodide symporter (NIS) mRNA expression. Previous studies showed that CpG-island methylation in the NIS proximal promoter does not correlate with mRNA expression.

OBJECTIVES

The aim of the study was to identify new CpG-islands within the NIS 5'region and investigate the involvement of their methylation in NIS expression.

DESIGN

DNA was obtained from 30 pairs of thyroid samples: tumor (T) and surrounding nontumor (NT) samples. All T samples had reduced NIS mRNA expression compared to NT samples.

MAIN OUTCOME MEASURES

Methylation degree was quantified by bisulfite sequencing, and NIS expression by real-time PCR and Western blot. Reporter gene assays were performed to determine CpG-island functionality. Tumor cell cultures were treated with 5-Aza demethylating agent to determine NIS expression, methylation status, and (125)I uptake.

RESULTS

We identified a new CpG-island2 with 14 CpG sites, located -2152/-1887 relative to ATG site. CpG-island2 was hypermethylated in T compared to NT samples, in both benign and malignant tumor groups. There was a significant inverse correlation between NIS mRNA expression and degree of CpG-island2 methylation in NT and T samples. This sequence increased the expression of a reporter gene; thus, it was considered a new enhancer. Cell culture treatments with 5-Aza reduced CpG-island2 methylation levels concomitantly with restoration of NIS mRNA and protein expression and (125)I uptake.

CONCLUSIONS

We identified a new distal enhancer, NIS distal enhancer, that regulates gene expression through DNA methylation. This enhancer is hypermethylated in T compared to NT samples and is associated with decreased NIS expression in tumors. This epigenetic deregulation may be an early event in tumorigenesis.