Epigenetic silencing of TTF-1/NKX2-1 through DNA hypermethylation and histone H3 modulation in thyroid carcinomas.
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 differentiation-inducing therapy through epigenetic modification.
Kondo
Tetsuo
T
Department of Pathology, University of Yamanashi, Yamanashi, Japan. ktetsuo@yamanashi.ac.jp
Nakazawa
Tadao
T
Ma
Defu
D
Niu
Dongfeng
D
Mochizuki
Kunio
K
Kawasaki
Tomonori
T
Nakamura
Nobuki
N
Yamane
Tetsu
T
Kobayashi
Makio
M
Katoh
Ryohei
R
D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D001483:Base Sequence; D002291:Carcinoma, Papillary / Q000209:etiology / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D018899:CpG Islands; D019175:DNA Methylation; D017931:DNA Primers / Q000235:genetics; D000077209:Decitabine; D044127:Epigenesis, Genetic / Q000187:drug effects; D020869:Gene Expression Profiling; D020868:Gene Silencing; D006657:Histones / Q000378:metabolism*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D009687:Nuclear Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D013961:Thyroid Gland / Q000378:metabolism; D013964:Thyroid Neoplasms / Q000209:etiology / Q000235:genetics* / Q000378:metabolism*; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors / Q000235:genetics*
D017931:DNA Primers; D006657:Histones; D006877:Hydroxamic Acids; C000614412:NKX2-1 protein, human; D009687:Nuclear Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors; C012589:trichostatin A; D000077209:Decitabine; D001374:Azacitidine