Disruption of cell-type-specific methylation at the Maspin gene promoter is frequently involved in undifferentiated thyroid cancers

Oncogene (2004) 23, 1117–1124 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $25.00 www.nature.com/onc Disruption of cell-type-specific methylation at the Maspin gene promoter is frequently involved in undifferentiated thyroid cancers Satoshi Ogasawara*,1,2,3, Chihaya Maesawa1, Masahide Yamamoto2, Yuji Akiyama2, Kei Wada1, Kentaro Fujisawa1, Taro Higuchi2, Yuki Tomisawa2, Nobuhiro Sato3, Shigeatsu Endo3, Kazuyoshi Saito2 and Tomoyuki Masuda1 1 Department of Pathology, Iwate Medical University School of Medicine, Uchimaru 19-1, 020-8505 Morioka, Japan; 2Department of Surgery 1, Iwate Medical University School of Medicine, Uchimaru 19-1, 020-8505 Morioka, Japan; 3Department of Critical Care Medicine, Iwate Medical University School of Medicine, Uchimaru 19-1, 020-8505 Morioka, Japan Cancer-associated DNA hypomethylation is as prevalent as cancer-linked hypermethylation, but the biological significance of DNA hypomethylation in carcinogenesis is less understood. The expression of Maspin (mammary serpin) in differentiated normal cells is regulated by epigenetic modifications in a cell-type-specific manner. Paradoxical Maspin expression due to epigenetic modification has been addressed in several cancer cell types. To elucidate the role of the Maspin gene in thyroid cancer, we studied methylation status in the promoter region and its expression in six human undifferentiated thyroid cancer cell lines and in specimens from 92 primary thyroid tumors, consisting of six follicular adenomas, 56 welldifferentiated thyroid cancers (WDTCs), 17 poorly differentiated thyroid cancers (PDTCs) and 13 undifferentiated thyroid cancers (UDTCs). Three of the six cell lines overexpressed Maspin mRNA and its protein product, but the remaining three did not. The methylation status at the promoter region was inversely correlated with Maspin expression. In Maspin-negative cell lines, Maspin expression was induced by treatment with 5-aza-20 deoxycytidine, a DNA demethylating agent. Immunoreactivity for Maspin protein was frequently detected in UDTCs (8/13, 62%) and PDTCs (7/17, 41%). Immunoreactivity for Maspin was diffusely positive in UDTCs, and was restricted to dedifferentiated components of the tumor in PDTCs. Positive immunoreactivity was infrequent in WDTCs (1/56, 2%), and all follicular adenomas and normal thyroid glands were completely negative. Their methylation status evaluated by the methylation-specific PCR method showed a good inverse correlation with their immunoreactivity in surgically resected specimens. Our data suggest that overexpression of Maspin by DNA hypomethylation is closely associated with morphological dedifferentiation in thyroid cancers. *Correspondence: S Ogasawara, Department of Surgery 1, Iwate Medical University School of Medicine, Uchimaru 19-1, 020-8505 Morioka, Japan; E-mail: Supported by Grant-in-Aid for Scientific Research 14571222, 14571221 and 15790698 from the Japanese Ministry of Education, Science, Sports and Culture Received 29 July 2003; revised 12 September 2003; accepted 15 September 2003 Oncogene (2004) 23, 1117–1124. doi:10.1038/sj.onc.1207211 Keywords: maspin; methylation; thyroid cancer; differentiation; cell-type-specific expression Introduction The 42-kDa protein Maspin (mammary serpin), a member of the serine protease inhibitor superfamily (Zou et al., 1994), is a unique tumor suppressor because of its biological behavior and function. It has recently been reported that Maspin expression is downregulated during progression of breast, prostate and oral carcinomas (Zou et al., 1994; Sheng et al., 1996; Xia et al., 2000). This finding is consistent with the fact that Maspin can act as a negative regulator of cell proliferation and survival, as cancer cells tend to have an increased proliferation rate and a reduced sensitivity to apoptosis (Jiang et al., 2002). Other recent investigations have demonstrated that frequent silencing of Maspin expression occurs in breast cancer cell lines (Domann et al., 2000; Futscher et al., 2002). In all cell lines in which Maspin expression was downregulated, Maspin promoter regions were hypermethylated and its expression was restored by treatment with a demethylating agent (Domann et al., 2000; Futscher et al., 2002). These findings support the notion that hypermethylation of CpG island loci influences cancer progression. However, a few reports have described that Maspin was overexpressed in pancreatic and ovarian cancers, and that Maspin transcripts were negative in the corresponding normal tissues (Maass et al., 2001; Sood et al., 2002). Thus, paradoxical Maspin protein expression due to epigenetic modification (hyper- or hypomethylation) has been described among various cancer cell types, making it difficult to understand the biological behavior of Maspin as a tumor suppressor. Futscher et al. (2002) have demonstrated that Maspin expression of normal cells is regulated by epigenetic modifications in a cell-type-specific manner. Their Maspin expression in thyroid cancer S Ogasawara et al 1118 report provides an important insight into the controversial issue of whether DNA methylation controls tissue-specific gene expression. Maspin-positive epithelial cells (airway, breast, skin and prostate) showed no methylation at CpG islands in the Maspin promoter region (Futscher et al., 2002). By contrast, Maspinnegative cells (skin fibroblast, lymphocytes, heart, liver and bone marrow) showed extensive methylation. Thus, cell-type-specific expression of Maspin is inversely correlated with the methylation status of the Maspin promoter (Futscher et al., 2002). This cell-type-specific regulation mechanism in differentiated cell types may contribute to paradoxical Maspin expression among cancers. It has been suggested that extensive DNA hypomethylation precedes cancer-linked DNA hypermethylation (Feinberg and Vogelstein, 1983; Gama-Sosa et al., 1983). DNA hypomethylation in cancer often affects more of the genome than does hypermethylation, so that net losses of genomic 5-methylcytosine are seen in many human cancers (Gama-Sosa et al., 1983). In an experimental analysis of DNA methyltransferase-deficient mice, Jaenisch and colleagues recently demonstrated that DNA hypomethylation plays a causal role in tumor formation, possibly by promoting chromosomal instability (Eden et al., 2003; Gaudet et al., 2003). The role of cancer-linked DNA hypomethylation is drawing more attention in cancer research (Ehrlich, 2002; Ehrlich et al., 2002). We recently reported that Maspin protein is selectively observed at high levels in gastric cancer cells as well as normal epithelium with intestinal metaplasia, but not in gastric normal epithelium without intestinal metaplasia (Akiyama et al., in press). Demethylation on the haploid allele of the Maspin promoter produces abundant protein expression in Maspin-positive gastric cancer cells and normal epithelium with intestinal metaplasia. Moreover, the extent of hypomethylation of the Ma (...truncated)