Epigenetic silencing of TTF-1/NKX2-1 through DNA hypermethylation and histone H3 modulation in thyroid carcinomas

Abstract

Thyroid transcription factor-1 (TTF-1), also known as NKX2-1, is a homeodomain containing transcriptional factor identified in thyroid, lung and central nervous system. In the thyroid, TTF-1 is essential for thyroid organogenesis and governs thyroid functions by regulating various thyroid-specific genes. We previously demonstrated that most differentiated thyroid neoplasms, including follicular adenomas/carcinomas and papillary carcinomas, express TTF-1 at both protein and mRNA levels. However, certain subtypes of thyroid cancers have shown low or negative expression of TTF-1. The aim of our study was to investigate the function of epigenetic modification in dysregulation of TTF-1 in thyroid carcinoma cells. We evaluated the expression of TTF-1 in primary thyroid tissues (normal thyroid, papillary carcinoma and undifferentiated carcinoma) and in thyroid carcinoma cell lines using immunohistochemistry and RT-PCR. Methylation-specific PCR targeting CpG islands of TTF-1 and chromatin immunoprecipitation (ChIP) for histone H3 lysine 9 (H3-lys9) were applied to clarify the correlation of the TTF-1 expression profile and epigenetic status. We also explored whether epigenetic modifiers, including 5-aza-deoxycytidine, could restore TTF-1 expression in thyroid carcinoma cells. In our current study, immunohistochemistry and RT-PCR showed positive expression of TTF-1 in normal thyroids and papillary carcinomas. Meanwhile, most of the undifferentiated carcinomas and the cell lines lost TTF-1 expression. No methylation in the CpG of TTF-1 promoter was detected in normal thyroids or papillary carcinomas. In contrast, DNA methylation was identified in 60% of the undifferentiated carcinomas (6/10) and 50% of the cell lines (4/8). ChIP assay demonstrated that acetylation of H3-lys9 was positively correlated with TTF-1 expression in thyroid carcinoma cells. Finally, DNA demethylating agents could restore TTF-1 gene expression in the thyroid carcinoma cell lines. Our data suggest that epigenetics is involved with inactivation of TTF-1 in thyroid carcinomas, and provide a possible means of using TTF-1 as a target for differentiationinducing therapy through epigenetic modification. Thyroid cancer is the most common malignancy of the endocrine organs, and its incidence rate has steadily increased over the last decade. 1 More than 95% of thyroid carcinomas are derived from follicular cells having a spectrum of differentiation from comparatively indolent carcinomas, including follicular thyroid carcinoma and papillary thyroid carcinoma (PTC), to the most invasive and lethal human malignancy, undifferentiated (anaplastic) thyroid carcinoma (UTC). 1 This wide spectrum of progression has been closely linked with a pattern of cumulative genetic defects that correlate with tumor differentiation, metastatic potential and aggressiveness. 2 Of these, RET rearrangements (RET/PTCs) and activating somatic mutations in RAS and BRAF oncogenes are mutually exclusive and represent important genetic events in thyroid carcinogenesis. [2][3][4] In addit...

Epigenetics of thyroid cancer and novel therapeutic targets

“…Promoter hypermethylation and silencing of the TSHR gene have been documented in thyroid carcinomas (Xing et al 2003, Hoque et al 2005, and similar findings have been reported for NIS and pendrin (Venkataraman et al 1999, Neumann et al 2004, Xing 2007. Furthermore, the expression of TTF1, a transcription factor that regulates the expression of all thyroid-specific genes (Bingle 1997), has been found to be silenced in poorly differentiated thyroid cancers as a result of promoter hypermethylation and histone H3 modifications (Kondo et al 2009). …”

“…As for the latter view, several lines of evidence indicate that epigenetic changes are early events in the tumorigenesis process, prompting Feinberg et al (2006) to propose an epigenetic progenitor origin of human cancer. The investigation of epigenetic mechanisms in cancer has been greatly facilitated by the development of the chromatin immunoprecipitation (ChIP) procedure, which has recently been used to obtain interesting data on the epigenetic status of human thyroid cancer cells (Kondo et al 2009). …”

Neural lineage-specific homeoprotein BRN2 is directly involved in TTF1 expression in small-cell lung cancer

“…It was recently reported that TTF1 expression deficiency in thyroid cancers is caused by epigenetic modification in association with cancer progression. 25 However, there have been no studies on TTF1 gene methylation in lung cancers.…”

Differentiation Therapy in Thyroid Carcinoma

“…Trichostatin A® acted pro-apoptotic and increased NIS mRNA expression in TC cell lines (Puppin et al, 2005;Shen & Chung, 2005;Kondo et al, 2009). mRNA expression of PDS was reduced by trichostatin A treatment (Zarnegar et al, 2002).…”

“…In a study on mRNA expression of NIS, Tg, TPO and TSH-R and on iodide uptake in the PTC cell line B-CPAP, 5-Azacytidine compared to ATRA and trichostatin was very effective: it was the only compound, which increased iodide uptake (Tuncel et al, 2007). Also 5-Aza-2'-deoxycytidine increased differentiation and restored NIS expression in FTC, PTC and ATC cell lines (Kondo et al, 2009). 5-Aza-2'-deoxycytidine also induced mRNA expression of type I iodothyronine-5'-deiodinase, another thyroid-specific protein (Mentrup et al, 2002 ).…”