Thymic epithelial tumors comprise thymoma, thymic carcinoma and neuroendocrine tumors of the thymus. Recent studies have revealed that the incidence of somatic non-synonymous mutations is significantly higher in thymic carcinoma than in thymoma. However, limited information is currently available on epigenetic alterations in these types of cancer. In this study, we thus performed genome-wide screening of aberrantly methylated CpG islands in thymoma and thymic carcinoma using Illumina HumanMethylation450 K BeadChip. We identified 92 CpG islands significantly hypermethylated in thymic carcinoma in relation to thymoma and selected G protein subunit gamma 4 (GNG4), growth hormone secretagogue receptor (GHSR), homeobox D9 (HOXD9) and spalt like transcription factor 3 (SALL3), which are related to cancer. We examined the promoter methylation of 4 genes in 46 thymic epithelial tumors and 20 paired thymus tissues using bisulfite pyrosequencing. Promoter methylation was significantly higher in thymic carcinoma than in thymoma and revealed a high discrimination between thymic carcinoma and thymoma in all 4 genes. Promoter methylation was higher in thymic carcinoma than in the thymus. No significant differences were observed in the promoter methylation of GNG4, HOXD9, or SALL3 between thymoma and the thymus. The promoter methylation of the 4 genes was not significantly higher in advanced-stage tumors than in early-stage tumors in all thymic epithelial tumors. Among the 4 genes, relapse-free survival was significantly worse in tumors with a higher DNA methylation than in those with a lower DNA methylation in all thymic epithelial tumors. Moreover, relapse-free survival was significantly worse in thymomas with a higher DNA methylation of HOXD9 and SALL3 than in those with a lower DNA methylation. On the whole, the findings of this study indicated that the promoter methylation of cancer-related genes was significantly higher in thymic carcinoma than in thymoma and the thymus. This is a common epigenetic alteration of high diagnostic value in thymic carcinoma and may be involved in the carcinogenesis of thymic carcinoma. However, epigenetic alterations in the 3 genes, apart from GHSR, are not involved in the tumorigenesis of thymoma.

Thymic epithelial tumors (TETs) comprise thymomas and thymic carcinoma (TC). TC has more aggressive features and a poorer prognosis than thymomas. Genetic and epigenetic alterations in thymomas and TC have been investigated in an attempt to identify novel target molecules for TC. In the present study, genome-wide screening was performed on aberrantly methylated CpG islands in thymomas and TC, and the glutamate decarboxylase 1 gene (GAD1) was identified as the 4th significantly hypermethylated CpG island in TC compared with thymomas. GAD1 catalyzes the production of γ-aminobutyric acid from L-glutamic acid. GAD1 expression is abundant in the brain but rare in other tissues, including the thymus. A total of 73 thymomas and 17 TC tissues were obtained from 90 patients who underwent surgery or biopsy at Tokushima University Hospital between 1990 and 2017. DNA methylation was examined by bisulfite pyrosequencing, and the mRNA and protein expression levels of GAD1 were analyzed using reverse transcription-quantitative PCR and immunohistochemistry, respectively. The DNA methylation levels of GAD1 were significantly higher in TC tissues than in the normal thymus and thymoma tissues, and GAD1 methylation exhibited high sensitivity and specificity for discriminating between TC and thymoma. The mRNA and protein expression levels of GAD1 were significantly higher in TC tissues than in thymomas. Patients with TET with high GAD1 DNA hypermethylation and high mRNA and protein expression levels had significantly shorter relapse-free survival rates than those with low levels. In conclusion, significantly more epigenetic alterations were observed in TC tissues compared with in thymomas, which may contribute to the clinical features and prognosis of patients.

Hexavalent chromium is recognized as a human carcinogen. Our previous studies revealed that lung cancer (LC) in chromate-exposed workers (chromate LC) had molecular features of frequent microsatellite instability (MSI), repression of MLH1 level, and aberrant DNA methylation of several tumor-suppressor genes, including MLH1. In the present study, we quantitatively investigated MLH1-promoter methylation status using bisulfite pyrosequencing of paired tumorous/nontumorous tissues from chromate and nonchromate LCs to determine the effect of chromate exposure on MLH1-promoter methylation. The methylation level of MLH1 promoter was significantly higher in chromate LC tumors (P < .001) than nonchromate LC tumors and, among chromate LC, significantly higher in tumorous tissue than nontumorous tissue (P = .004). Moreover, the methylation level of MLH1 promoter in normal lung tissue tended to be higher in chromate LC than nonchromate LC (P = .062). In addition, LC with reduced levels of MLH1 showed significantly higher methylation levels of MLH1 promoter than LC exhibiting normal MLH1 levels (P = .019). Moreover, immunohistochemical analyses determined that levels of SUV39H1, an H3K9me2-related methyltransferase, were higher in chromate LC than nonchromate LC (P = .076). Furthermore, we evaluated three DNA double-strand break-repair genes (MRE11, RAD50, and DNA-PKcs) as possible targets of MSI by fragment-length polymorphism analysis, revealing the mutation frequency of RAD50 as significantly higher in chromate LC than nonchromate LC (P = .047). These results suggest that chromate exposure might induce MLH1 hypermethylation in LC as a mechanism of chromate-induced carcinogenesis.

Our previous study reported that the DNA methylation of growth hormone secretagogue receptor ( GHSR ) was significantly higher in thymoma or thymic carcinoma (TC) than in normal thymic tissue samples. Thymic epithelial tumors (TETs) with higher GHSR DNA methylation were associated with significantly worse prognosis than those with lower levels of DNA methylation. Diversified components of the ghrelin- GHSR axis may exert opposing effects in cancer progression, depending on the cancer type in question. However, the precise function of the axis remains unclear. In the present study, the mRNA expression of five key components of the ghrelin system [native ligand ghrelin, variant ligand In-1 ghrelin, native receptor GHSR1a, variant receptor GHSR1b and acylation enzyme ghrelin O-acyltransferase (GOAT)] were examined in 58 TET samples by reverse transcription-quantitative PCR, and protein expression of GHSR1a and GHSR1b was assessed in 20 TETs using immunohistochemistry. The results revealed that In-1 ghrelin, GHSR1b (variant forms) and GOAT were more strongly expressed in thymoma compared with thymic-adjacent tissue. By contrast, no significant differences were observed in the expression of ghrelin and GHSR1a (native forms) between thymoma and thymic tissue. The mRNA expression of In-1 ghrelin and GHSR1b (variant forms) was positively associated with GHSR methylation in thymoma tissue samples. However, a relationship was not found between ghrelin, GHSR1a or GOAT expression (native forms) and GHSR methylation in thymoma. Immunohistochemical analysis revealed that mRNA expression of GHSR1a and GHSR1b generally correlated with expression of the corresponding protein, and that the expression of GHSR1b was increased in advanced-stage TETs. These results indicate that the DNA methylation of GHSR is associated with a shift from native expression (ghrelin and GHSR1a) to variant expression (In-1 ghrelin and GHSR1b), which induces the tumorigenesis of thymoma, but not TC.

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