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. 2013 Jan 31;32(5):663-9.
doi: 10.1038/onc.2012.67. Epub 2012 Mar 5.

Tumor development is associated with decrease of TET gene expression and 5-methylcytosine hydroxylation

H Yang  1 , Y Liu, F Bai, J-Y Zhang, S-H Ma, J Liu, Z-D Xu, H-G Zhu, Z-Q Ling, D Ye, K-L Guan, Y Xiong
Affiliations

Tumor development is associated with decrease of TET gene expression and 5-methylcytosine hydroxylation

H Yang et al. Oncogene. .

Abstract

The TET (ten-eleven translocation) family of α-ketoglutarate (α-KG)-dependent dioxygenases catalyzes the sequential oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine and 5-carboxylcytosine, leading to eventual DNA demethylation. The TET2 gene is a bona fide tumor suppressor frequently mutated in leukemia, and TET enzyme activity is inhibited in IDH1/2-mutated tumors by the oncometabolite 2-hydroxyglutarate, an antagonist of α-KG, linking 5mC oxidation to cancer development. We report here that the levels of 5hmC are dramatically reduced in human breast, liver, lung, pancreatic and prostate cancers when compared with the matched surrounding normal tissues. Associated with the 5hmC decrease is the substantial reduction of the expression of all three TET genes, revealing a possible mechanism for the reduced 5hmC in cancer cells. The decrease of 5hmC was also observed during tumor development in different genetically engineered mouse models. Together, our results identify 5hmC as a biomarker whose decrease is broadly and tightly associated with tumor development.

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Conflict of interest statement

Conflict of interest

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
5hmC is substantially reduced in multiple human tumors. IHC was performed using antibodies against 5hmC and Ki67 in human normal and cancer tissues from liver, breast, lung and pancreas (a). The mean values of the IHC quantification are shown (b). Scale bars are 20 µm. Data are represented as mean±s.d. Freshly frozen or paraffin-embedded human normal tissue and tumors of liver, breast, lung, prostate and pancreas were acquired from Department of Pathology, Huashan Hospital, Fudan University. The procedures relating to the acquisition of samples from human subjects were approved by the Ethics Committee of the Institutes of Biomedical Sciences (IBS), Fudan University. The paraffin-embedded specimens were cut into 5 µm thin sections, which were subsequently stained with hematoxylin and eosin (H/E). Subsequent separation of tumor and normal surrounding tissues were carried out microscopically by an experienced molecular pathologist and guided by the H/E staining. For IHC analysis, the labeled streptavidin-biotin (LSAB) method was applied using commercial kits (Dako Corporation, Santa Barbara, CA, USA). Briefly, paraffin sections were deparaffinized and rehydrated following standard protocols. Sections were incubated with 3% H2O2 to eliminate the endogenous peroxidase activity, and then blocked with 5% normal goat serum. After treatment with 2N HCl for 15 min at room temperature, sections were neutralized with 100mm Tris–HCl (pH 8.5) for 10 min and then washed three times with PBS, followed by incubation with a primary anti-5hmC antibody (Active Motif; Cat. 39769, dilution at 1:1000) at 37 °C for 1 h. A horseradish peroxidase (HRP)-conjugated secondary antibody (Dako Corporation) was then applied and incubated at 37 °C for 1 h. For Ki67 IHC staining, the LSAB method was used as described above, using a primary anti-Ki67 antibody (dilution 1:100; Abcam, Cambridge, MA, USA) and a HRP-conjugated secondary antibody (Dako Corporation). Sections were developed with DAB kit and stopped with water. To semiquantify the 5hmC-positive areas, five randomly selected fields (173 µm2 each) from each sample were randomly selected and microscopically examined. Images were captured using a charge-coupled device (CCD) camera and analyzed using Motic Images Advanced software (version 3.2, Motic China Group Co. Ltd). The relative 5hmC intensity was calculated by dividing the positively stained areas over the total area. All IHC image analysis values were calculated as mean ± s.d. Statistical analysis was performed using SPSS 11.5 statistical package. Statistical methods included Paired t-test and independent samples t-test. All statistical tests were considered significant at an α=0.05 (P < 0.05).
Figure 2
Figure 2
5hmC levels are reduced in mouse tumors. IHC was performed using antibodies against 5hmC and Ki67 in two types of mouse tumors developed in different genetic backgrounds, a lymphoma invaded in liver and a prostate tumor, and counterstained with hematoxylin (a). The 5hmC IHC quantification is shown (b). Scale bars are 100 µm. Data are represented as mean ± s.d. (n=5).
Figure 3
Figure 3
Quantification of 5hmC decrease in human breast cancer. (a) To validate the dot-blot assay, the anti-5hmC antibody was preincubated with equal amount (1mm) of dhmCTP, dmCTP or dCTP for 1 h at room temperature, and then was used for dot-blot hybridization. Note that the anti-5hmC antibody very specifically recognized 5hmC, and could be effectively blocked by the preincubation with dhmCTP, but not dmCTP or dCTP. In addition, the dot-blot membrane was hybridized with 0.02% methylene blue in 0.3M sodium acetate (pH 5.2) to stain DNA. (b) Genomic DNAs were extracted from 15 pairs of paraffin-embedded human breast carcinoma and the matched normal breast tissues. In 12 of the 15 paired samples, sufficient genomic DNA was harvested for dot-blot hybridization. Representative micrographs of dot-blot assay, 5hmC and Ki67 staining in four paired samples are shown. Dot-blot results of the other eight pairs of normal and cancer samples are shown in Supplemental Figure S3. (c) Genomic DNAs were extracted from five pairs of paraffin-embedded human lung carcinoma and the matched normal lung tissues and subjected to dot-blot hybridization to determine the levels of 5hmC. Genomic DNAs were extracted from paraffin-embedded sections from normal and tumor samples following standard protocols. In detail, paraffin-embedded sections (10 ìm thickness) were deparaffinized by xylene. The deparaffinized samples were treated with ribonuclease A (50 µg/ml; Takara) at 37 °C for 1 h, and then incubated with proteinase K (0.3–0.5 mg/ml, Takara) in a digestion buffer (100mm NaCl/10mm Tris–HCl, pH 8.0 and 25mm EDTA, pH 8.0/0.5% SDS) at 55 °C overnight. After complete RNase and proteases digestion, genomic DNA was extracted with the same volume of phenol/chloroform/isoamyl alcohol (25:24:1) (Sangon, Shanghai), and then precipitated with an equal volume of isopropanol. After centrifugation, DNA pellet was washed once with 75% ethanol, air dried and dissolved in 15 µl distilled water. The DNA concentration was measured by NanoDrop (Thermo Scientific). The procedure for the dot-blot assay was modified from a procedure described previously (Xu et al., 2011). Briefly, DNA was spotted on a nitrocellulose membrane (Whatman), which was placed under an ultraviolet lamp for 20 min to crosslink the DNA. Subsequently, the membrane was blocked with 5% milk in TBS-Tween for 1 h and incubated with the primary anti-5hmC antibody at 4 °C overnight. After incubation with a horse radish peroxidase-conjugated secondary antibody anti-rabbit IgG (GenScript) for 1 h at room temperature, the membrane was washed with TBS-Tween 20 for three times and then detected and scanned by a Typhoon scanner (GE Healthcare). The 5hmC intensity was quantified by Image-Quanta software (GE Healthcare).
Figure 4
Figure 4
Reduced 5hmC is associated with the substantial reduction of TET gene expression in human cancers. Genomic DNA and mRNA were extracted from three pairs of frozen human breast carcinoma and the matched normal breast tissues (a) and three pairs of frozen human HCC and the matched normal liver tissues (b). The levels of 5hmC and mRNAs of three TET genes were determined by dot-blot assay and quantitative real-time PCR, respectively. Data are represented as mean ± s.d. RNA was extracted using QIAamp RNA Mini Kit following the manufacturer’s instructions (Qiagen). Quantitative real-time PCR was performed using an Applied Biosystems 7500 Sequence Detection System with SYBR green labeling with β-actin as an endogenous control. Primer sequences are listed in Supplementary Table S1.
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