BACKGROUND : Regulator of Calcineurin 1.4 (RCAN1.4) is a functionally downregulated metastasis progression suppressor (MPS) in thyroid cancer; however, the mechanisms for RCAN1.4 loss in thyroid cancer have not yet been reported. The RCAN1.4 promoter and gene contain several CG-rich regions, some of which are reported to be hypermethylated in non-thyroid tissues. We therefore hypothesized that RCAN1.4 downregulation in thyroid cancer was in part due to hypermethylation.

METHODS : Studies were performed in 5 thyroid cancer cell lines (TPC1, FTC133, BCPAP, C643, and 8505C) with different genetic drivers, and in 18 paired normal and thyroid cancer human thyroid cancer tissues. Basal RCAN1.4 mRNA and protein levels were assessed in all of the cell lines. Cell lines with lowest RCAN 1.4 expression levels were treated with the DNA methyl transferase inhibitor, Decitabine. Normal/Tumor tissue pairs were analyzed for methylation of three CG-rich regions both by capture of methylated DNA by MBD2 protein and by methylation specific PCR (MSPCR).

RESULTS : In all assessed cell lines, RCAN1.4 mRNA and protein levels increased following Decitabine treatment. In silico analysis of the RCAN1.4 gene identified three CG rich regions as possible methylation targets; one in the proximal promoter and two in intron 1. Hypermethylation of the intron 1 CG rich regions was identified by both the capture method and MSPCR. In contrast, there was hypermethylation of the CG rich region of the proximal promoter was not identified. Gene expression confirmed that hypermethylation in thyroid cancer samples in intron1 of RCAN1.4 was associated with lower levels of RCAN1.4 mRNA. Finally, the cancer samples demonstrated increased NFE2L3 expression, a downstream marker of functional RCAN1.4 loss.

CONCLUSIONS : The MPS gene, RCAN1.4, is downregulated in thyroid cancer cells and human thyroid cancer in part by hypermethylation of CG rich regions in intron 1.  .