4502

Thyroid cancer cells often do not express thyroid-related genes including sodium iodide symporter (NIS), thyroperoxidase (TPO), thyroglobulin (TG) and thyrotrophin-stimulating hormone receptor (TSHR); therefore, these cells are thought to be a poorly differentiated population. Analysis of up-stream regulator(s) of these genes is one of the strategies for investigating abnormalities of thyroid cancer.

Database analysis revealed that the promoter region of NIS, TPO, TG and TSHR genes contain common transcription factor binding sites including HNF3β/FoxA2, C/EBP, Pax-8, TTF-1 and PPARγ. Quantitative RT-PCR showed that the mRNA expression of HNF3β/FoxA2, C/EBPβ, and TTF-1 was markedly decreased in five thyroid cancer cell lines compared to normal thyroid cells; the expression levels of HNF3β/FoxA2, C/EBPβ, and TTF-1 in normal thyroid cells were 12.3-fold, 1.9-fold and 21.3-fold higher than thyroid cancer cell lines, respectively. Interestingly, the HNF3β/FoxA2 gene has a CpG island in its promoter region; therefore, we analyzed the methylation status in the region. Sodium bisulfate conversion of genomic DNA followed by PCR analysis showed that the gene is methylated in the BHP 2-7 papillary thyroid cancer cell line but not in the normal thyroid cells. In addition, the expression of HNF3β/FoxA2 mRNA was induced in the cells after their treatment with 5-aza-2-deoxycytidine, an inhibitor of DNA methyltransferase. These results suggest that the HNF3β/FoxA2 gene is methylated and silenced in some thyroid cancers.

We also examined the cellular localization of C/EBPβ; to behave as a transcription factor, it requires nuclear localization. Immunohistochemical staining showed that it is localized in the nucleus in normal thyroid cells; in contrast, the protein is detected in the cytoplasm in papillary thyroid cancer cells. Subcellular fractionation of BHP 2-7 papillary thyroid cancer cell line followed by Western blot analysis showed high level of expression of C/EBPβ in the cytoplasm, suggesting that a large proportion of C/EBPβ protein cannot translocate into the nucleus and function as a transcription factor in papillary thyroid cancer cells.

Taken together, these observations indicate two clear abnormalities of transcription factors in thyroid cancer cells: abnormal methylation of HNF3β/FoxA2 and inappropriate cellular localization of C/EBPβ.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA

American Association for Cancer Research
2007