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.
Genomic DNA methylation, occurring predominantly on the 5th carbon atom of cytosine (5-methylcytosine (5mC)) in the context of CpG islands in mammals, has a broad and important role in normal development and tumor suppression (Jones and Baylin, 2007). The enzymes that catalyze DNA methylation, DNA methyltransferases, have been thoroughly studied, while the enzymes and mechanisms of DNA demethylation have remained elusive, especially in animals (Wu and Zhang, 2010). A recently discovered family of Fe(II)- and α-ketoglutarate (α-KG)-dependent dioxygenases, the TET (ten–eleven translocation) proteins, has the capacity to catalyze a three-sequential oxidation reactions: converting 5mC first to 5-hydroxymethylcytosine (5hmC), and then 5-formylcytosine, and finally 5-carboxylcytosine (5caC) (Tahiliani et al., 2009; Ito et al., 2010, 2011; He et al., 2011). A subsequent decarboxylation of 5caC, by either a thymine-DNA glycosylase or other DNA repair enzymes, could then lead to DNA demethylation.
Mammalian cells express three TET genes: TET1, TET2 and TET3. TET2 is mutationally inactivated in about 15% of myeloid cancers, including 22% of acute myeloid leukemia (AML) (Delhommeau et al., 2009; Langemeijer et al., 2009). Separately, AML (Mardis et al., 2009), as well glioma (Parsons et al., 2008), chondrosarcoma (Amary et al., 2011a), enchondroma (Pansuriya et al., 2011; Amary et al., 2011b), thyroid carcinomas (Hemerly et al., 2010; Murugan et al., 2010) also sustain frequent mutations in genes encoding for the metabolic enzymes isocitrate dehydrogenase (IDH1 and IDH2). IDH1 and IDH2 mutations have also been reported in a limited number of samples of several additional types of tumors at lower frequency. These mutations result in simultaneous loss and gain of activities in the production of α-KG and 2-hydroxyglutarate (2-HG), respectively (Dang et al., 2009; Zhao et al., 2009). 2-HG functions as an α-KG antagonist by binding to the same space in the catalytic site and competitively inhibiting the activity of α-KG-dependent dioxygenases, including TETs (Xu et al., 2011). These results provide a mechanistic interpretation for the mutually exclusive mutation patterns between IDH1/2 and TET2 genes in AML (Figueroa et al., 2010) and increased genomic methylation in IDH1-mutated gliomas (Noushmehr et al., 2010). They also highlight the important role of deregulated TET activity in tumorigenesis. Given these intrinsic links between decreased function of TET-catalyzed 5mC-to-5hmC conversion and tumorigenesis in both glioma and leukemia, we set to determine how broadly 5hmC decrease occurs in human tumors.
Results and discussion
5hmC is decreased in multiple human cancers
Immunohistochemistry (IHC) offers an efficient way for simultaneously examining the expression of a given antigen and histopathology of multiple samples in minimal quantity such as paraffin-embedded tissue sections. One critical reagent for reliable IHC is the quality of the antibody. To this end, we first verified the specificity of a polyclonal anti-5hmC antibody by antigen competition experiments. Using this antibody, we detected strong nuclear signals in normal human liver tissue, which were significantly diminished by preincubation of the antibody with the antigen dhmCTP (deoxyhydroxymethylcytidine triphosphate), but not by a similar preincubation with either dmCTP (deoxymethylcytidine triphosphate) or dCTP (deoxycytidine triphosphate) (Supplementary Figure S1). We then determined the levels and distribution of 5hmC in multiple types of human normal and cancer tissues, including liver, breast, lung, pancreas (Figure 1a), and prostate (Supplementary Figure S2). For liver samples, we assessed 11 normal liver (from liver transplantation), 44 peripheral liver, 10 liver adenoma and 49 HCC (hepatocellular carcinoma) samples. As shown normal liver and peripheral liver tissues displayed similar 5hmC levels. Notably, a significant decrease of 5hmC was observed in both liver adenoma and HCC as compared with normal liver and quantified via IHC (P<0.01) (Figure 1b). In human breast tissue, we evaluated a total of 15 pairs of normal and carcinoma samples. Again, we found that the levels of 5hmC were dramatically reduced in human breast cancer when compared with the matched surrounding normal breast tissues (P<0.01) (Figure 1b). In human lung, we evaluated a total of 39 samples and observed a profound reduction of 5hmC in the tumor samples when compared with normal lung tissues (P<0.01) (Figure 1b). In human pancreas, we examined 16 cases and observed a similar reduction of 5hmC in both types of tumors (P<0.01) (Figure 1b). Similar 5hmC reduction was also observed in human prostate tumors (P<0.01) (Supplementary Figure S2). To verify the 5hmC staining in tumor samples, we performed IHC staining to detect the Ki67 protein, a cellular marker for proliferation, in the corresponding sections of normal and tumor samples. As compared with normal tissues, the Ki67 signal is stronger in tumor sections where the 5hmC signal is weak (Figure 1a). This provides evidence supporting that all the tumor samples have been properly prepared. Together, our IHC staining data demonstrate that the levels of 5hmC are dramatically decreased during human tumor development independently of cancer types.
5hmC is decreased in different genetically engineered mouse models
To determine whether a similar 5hmC reduction also occurs during mouse tumor development and whether the decrease is independent of specific genetic alterations, we performed IHC staining and determined the 5hmC levels in tumor generated from different genetically engineered mouse models that we have previously characterized including invasive lymphoma in the livers of Eμ-Myc;Cul9+/− mice, and lung tumors in p18Ink4c;Brca1+/−. In both types of mouse tumors, we found that 5hmC was significantly (P-value) decreased (Figure 2), supporting the notion that the reduction of 5hmC is a property common to different tumors in both humans and mice.
Development of a dot-blot hybridization method for sensitive and quantitative analysis of 5hmC
Although IHC can determine the tissue distribution of an antigen alone with histopathological examination, it offers only a semiquantitative measurement. Taking advantage that 5hmC is an antigen present in DNA, we developed a simple, sensitive and quantitative assay based on dot-blot hybridization for determining the levels of 5hmC. We first evaluated the specificity of the anti-5hmC antibody for the dot-blot by antigen nucleotide blocking experiments. We found that the anti-5hmC antibody could detect a clear signal in as little as 50 ng genomic DNA isolated from normal human breast tissue by dot-blot assay. Preincubation of the anti-5hmC antibody with the antigen dhmCTP, but not d5mC or dCTP, completely blocked the signal recognized by this antibody (Figure 3a). Prompted by this sensitivity and specificity of the antibody, we tested the feasibility of determining 5hmC by dot-blot assay using genomic DNA isolated from paraffin-embedded tissue sections. We found that as few as five sections (10 μm) of normal breast tissues could provide sufficient genomic DNA for the detection of 5hmC by the dot-blot assay. In 12 of 15 pairs of breast tissue samples, sufficient genomic DNA was harvested for dot-blot analysis. Most importantly, in all 12 paired samples that were examined, the levels of 5hmC were strikingly decreased even when the highest amount of DNA was used (250 ng; Figure 3b). In addition, Ki67 IHC staining results suggest that these breast cancer samples have been properly prepared (Figure 3b). An accurate determination on how many folds of reduction of 5hmC was exhibited in breast carcinoma samples was not reliable due to the very low levels of 5hmC in tumor genomic DNA, but the level of 5hmC in every tumor sample was at least ten fold lower than the matched normal tissue. Likewise, using this dot-blot assay, we also found that 5hmC is substantially reduced in human lung tumors when compared with their matched normal surrounding tissues (Figure 3c). Together, the IHC and dot-blot assay demonstrate that 5hmC levels are significantly reduced in multiple human tumors when compared with their matched normal surrounding tissues.
5hmC decrease is associated with a substantial reduction of TET gene expression in human tumors
Two mechanisms have been reported that would lead to decreased levels of 5hmC in human tumors—loss-of-functional mutations targeting TET2 gene and inhibition of TET activity by the reduction of α-KG and accumulation of 2-HG resulting from IDH1/2 mutations (Delhommeau et al., 2009; Mullighan, 2009; Figueroa et al., 2010; Xu et al., 2011). However, neither TET nor IDH1/2 gene has thus far been reported to be mutated in liver, breast, lung, pancreas and prostate tumors. To investigate the possible mechanism underlying 5hmC decrease in these cancers, we determined the mRNA expression of three TET genes in both human breast and liver cancers. Interestingly, we found that the expressions of all three TET genes were significantly and uniformly reduced in both types of human tumors when compared with their matched normal tissues (Figures 4a and b). Of note, the decrease of three TET genes varied, with TET1 reduced most significantly, followed by TET2 and TET3. These observations provide a potential molecular mechanism for the observed reduction of 5hmC in human cancers.
In this paper, we demonstrate that 5hmC, a newly discovered modification of genomic DNA whose level is inversely correlated with that of 5mC, is substantially reduced in multiple types of human cancer. During the preparation of this paper, Haffner et al. (2011) reported that that 5hmC is substantially decreased in three types of human cancers, including breast, colon and prostate. Our study supports the link between 5hmC reduction and tumor development by expanding the finding to three additional types of cancer; liver, lung and pancreases. In every sample examined, remarkably, the tumor cells always have reduced 5hmC than the corresponding normal cells with no exception. Together, these studies suggest that 5hmC reduction is broadly and tightly linked with tumorigenesis. Moreover, our study shows that detection of 5hmC could be valuable biomarker for diagnosis of many cancer types. The tight inverse correlation between 5hmC and tumorigenesis suggests a potentially fundamental role of 5hmC and cytosine epigenetic modification in cancer development.
Our study also provides novel insights into the decrease of 5hmC levels in human tumors. First, we found that TET gene expression is significantly reduced in the tumors we examined, providing a potential mechanism underlying the 5hmC reduction. This result indicates that beside mutations targeting either TET2 or IDH1/2 genes, there are additional mechanisms, such as transcriptional inactivation of TET gene expression, which can block the 5mC-to-5hmC conversion and thus DNA demethylation. It will be important to determine how TET gene expression is silenced broadly in so many different types of tumors. Second, we showed that in different mouse tumor models that 5hmC was also decreased, indicating a gene-independent 5hmC reduction during tumor development. Lastly and importantly, we have developed a sensitive, reliable and quantitative dot-blot assay for determining the change in 5hmC levels using genomic DNA isolated from paraffin-embedded tissue sections. This assay should have broad applicability for clinical diagnosis.
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We thank the members of the Fudan MCB laboratory for discussions and support throughout this study, and Eric Oermann for reading the manuscript. This work was supported by MOST 973 (No. 2009CB918401, No. 2011CB910600), NSFC (Grant No. 30600112, 30871255, 31071192). This work was also supported by the 985 Program, Shanghai key project (Grant No. 09JC1402300), the Shanghai Leading Academic Discipline Project (project number B110), and NIH grants (to YX and KLG).
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Oncogene website
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Yang, H., Liu, Y., Bai, F. et al. Tumor development is associated with decrease of TET gene expression and 5-methylcytosine hydroxylation. Oncogene 32, 663–669 (2013). https://doi.org/10.1038/onc.2012.67
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