Frequent epigenetic silencing of the CpG island promoter of RASSF1A in thyroid carcinoma.

Loss of heterozygosity of chromosome 3p21 is one of the most frequent alterations in solid tumors, including thyroid carcinomas. Recently, we have characterized the novel tumor suppressor gene RASSF1 located in this locus. The RASSF1A isoform is epigenetically inactivated in a variety of human primary tumors. In this study, we investigated the expression and methylation status of the RASSF1 gene in thyroid carcinoma. In nine thyroid cancer cell lines, the RASSF1A promoter CpG island was methylated completely, and expression was absent. Treatment of these cell lines with the DNA methylation inhibitor 5-aza-2'-deoxycytidine reactivated the transcription of RASSF1A. The methylation status of the RASSF1A promoter was analyzed in 38 primary thyroid tumors, including 1 poorly differentiated thyroid carcinoma, 5 medullary thyroid carcinoma (MTC), 10 follicular thyroid carcinoma (FTC), 9 undifferentiated thyroid carcinoma (UTC), and 13 papillary thyroid carcinoma (PTC). In 71% of thyroid carcinomas, the RASSF1A CpG island was hypermethylated. Methylation frequency was higher in the aggressive forms of thyroid carcinoma and was found in 80% of MTC, in 78% of UTC, and in 70% of FTC, compared with 62% in the more benign PTC. RASSF1A inactivation was detected in all stages of thyroid carcinoma scored by Tumor-Node-Metastasis classification. Additionally, we analyzed the methylation frequency of the CpG island of cell cycle inhibitor p16(INK4a) in the same thyroid tumors. The p16 gene was inactivated in 56 and 25% of cell lines and primary tumors, respectively. p16 methylation was detected in 56% of UTC, 10% of FTC, and 25% of PTC but not in MTC. In UTC, which belongs to the most aggressive carcinomas in humans, the most common combined inactivation of RASSF1A and p16 was detected. In general, 90% of tumors with p16 inactivation were also silenced for RASSF1A expression. However, RASSF1A hypermethylation was detected three times more frequently in thyroid cancers. Thus, RASSF1A inactivation may play a crucial role in the malignancy of thyroid carcinoma.