Aberrantly methylated genes in human papillary thyroid cancer and their association with BRAF/RAS mutation

Abstract

Cancer arises through accumulation of epigenetic and genetic alteration. Aberrant promoter methylation is a common epigenetic mechanism of gene silencing in cancer cells. We here performed genome-wide analysis of DNA methylation of promoter regions by Infinium HumanMethylation27 BeadChip, using 14 clinical papillary thyroid cancer samples and 10 normal thyroid samples. Among the 14 papillary cancer cases, 11 showed frequent aberrant methylation, but the other three cases showed no aberrant methylation at all. Distribution of the hypermethylation among cancer samples was non-random, which implied existence of a subset of preferentially methylated papillary thyroid cancer. Among 25 frequently methylated genes, methylation status of six genes (HIST1H3J, POU4F2, SHOX2, PHKG2, TLX3, HOXA7) was validated quantitatively by pyrosequencing. Epigenetic silencing of these genes in methylated papillary thyroid cancer cell lines was confirmed by gene re-expression following treatment with 5-aza-2'-deoxycytidine and trichostatin A, and detected by real-time RT-PCR. Methylation of these six genes was validated by analysis of additional 20 papillary thyroid cancer and 10 normal samples. Among the 34 cancer samples in total, 26 cancer samples with preferential methylation were significantly associated with mutation of BRAF/RAS oncogene (P = 0.04, Fisher's exact test). Thus, we identified new genes with frequent epigenetic hypermethylation in papillary thyroid cancer, two subsets of either preferentially methylated or hardly methylated papillary thyroid cancer, with a concomitant occurrence of oncogene mutation and gene methylation. These hypermethylated genes may constitute potential biomarkers for papillary thyroid cancer.

Keywords: BRAF; CIMP (CpG island methylator phenotype); DNA methylation; RAS; oncogene mutation; thyroid cancer.

Figure 1

Representative data obtained from MALDI-TOF-MS imaging for three cancer samples. Cancer samples #1, #4, and #5 correspond to papillary cancer samples #1, #4, and #5 in Figure 3. First column: photomicrograph of a tumor section with hematoxylin/eosin (H/E) staining. Second, third, and fourth columns: MALDI-TOF-MS profile with detection of BRAF/RAS mutations. X-axis indicates mass-to-charge ratio (m/z) to distinguish wild-type and mutant allele, and Y-axis indicates signal intensity.

Figure 2

Heatmap for methylation status of several cancer types. Genes with β-value <0.17 in normal samples and with standard deviation of β-value in cancer samples >0.15, were selected and their β-values were shown by heatmap. Each cancer type showed different methylation profile, and papillary thyroid cancer displayed fewer aberrantly methylated genes than other cancer types. HNSCC, head and neck squamous cell carcinoma; CRC, colorectal cancer; PC, prostate cancer; CML, chronic myeloid leukemia; GC, gastric cancer; GBM, glioblastoma; PTC, papillary thyroid cancer; N, normal samples corresponding to these cancer types.

Figure 3

Aberrantly methylated genes frequently observed in papillary thyroid cancer. PBC, peripheral blood cells. We detected 25 genes that were hypermethylated (β > 0.25) in at least three of 14 papillary thyroid cancer samples, but not methylated (β < 0.2) in 10 normal samples. Among the 14 papillary cancer samples, 11 showed aberrant methylation in three or more genes, whereas the other three samples had no aberrant methylation. Purple, tumor size; brown, number of metastatic lymph nodes >3; gray, distant metastasis(+); green, age over 45 years old; pink, female; pale blue, stage III or IV; red, recurrence (+); orange, BRAF mutation(+); blue, RAS mutation (+). The two samples with recurrence (red) had no aberrant methylation (P = 0.03, Fisher's exact test). BRAF/RAS oncogene mutations were all observed in methylation(+) samples (P = 0.03, Fisher's exact test).

Figure 4

Confirmation of aberrant methylation by pyrosequencing. Six genes were randomly chosen among the 25 frequently methylated genes, and their methylation status was quantitatively validated by pyrosequencing, using the 14 papillary thyroid cancer samples and 10 normal thyroid samples that were analyzed by Infinium. Although one normal sample showed high methylation in POU4F2, aberrant methylation of the six genes in the papillary cancer samples was confirmed. Bottom, the corresponding data for aberrant methylation obtained by Infinium analysis (Figure 3).

Figure 5

Silencing of aberrantly methylated genes. (A) Methylation status of the HOXA7, POU4F2, HIST1H3J, TLX3, PHKG2, SHOX2 genes was analyzed in papillary thyroid cancer cell lines TPC1, KTC1, and KTC3 and anaplastic thyroid cancer cell line BHT-101 by pyrosequencing. Open box, 0–30% methylation, presumably no allele methylation. Hatched box, 30–70% methylation, presumably hemi-allelic or partial methylation. Closed box, 70–100% methylation, presumably bi-allelic and dense methylation. All the genes except SHOX2 showed dense methylation in at least one papillary thyroid cancer cell line. (B) Real-time RT-PCR analysis of the methylated genes. Cells were treated with 5-aza-2′-deoxycytidine (5-AZA) and/or trichostatin A (TSA). Gene expression levels were normalized to that of PPIA (Peptidylprolyl Isomerase A, or cyclophilin A). All the genes except SHOX2 showed no or very low expression in the analyzed cancer cell line, and showed re-expression in cells treated with 5-AZA/TSA.

Figure 6

Aberrant methylation of HOXA7, POU4F2, HIST1H3J, TLX3, PHKG2, SHOX2 genes in additional samples: 20 cancer samples and 10 normal ones. Methylation status of the six genes was confirmed by pyrosequencing. Bottom, color scale with known methylation degrees (0–100%).

Figure 7

Methylation status of the six genes in 54 samples analyzed by pyrosequencing. (A) Each gene was methylated to a different extent in each sample. C, 34 cancer samples. N, 20 normal samples. Circle, methylation ratio of each sample. Filled circles indicate that the gene is methylated with the methylation ratio >25%. Bars, the mean and standard error of methylation ratios. P-values were calculated by t-test to compare distribution of methylation ratios between cancer (C) and normal (N) samples. Five genes (HIST1H3J, SHOX2, PHKG2, TLX3, and HOXA7) showed significantly higher methylation in cancer (P < 0.05, t-test). POU4F2 tended to show higher methylation in cancer (P = 0.07, t-test). (B) Heatmap for methylation status of the 54 analyzed samples. Among the 34 papillary thyroid cancer samples, 26 were aberrant methylation(+) in at least one of six genes, while eight had no methylation. In Table 4, the number of cancer samples with aberrant methylation (n = 26) and the number of samples without aberrant methylation (n = 8) refer to these data.