Background Genetic aberrations in DNA repair genes are linked to cancer, but less is reported about epigenetic regulation of DNA repair and functional consequences. We investigated the intragenic methylation loss at the three prime repair exonuclease 2 ( TREX2 ) locus in laryngeal ( n = 256) and colorectal cancer cases ( n = 95) and in pan-cancer data from The Cancer Genome Atlas (TCGA). Results Significant methylation loss at an intragenic site of TREX2 was a frequent trait in both patient cohorts ( p = 0.016 and < 0.001, respectively) and in 15 out of 22 TCGA studies. Methylation loss correlated with immunohistochemically staining for TREX2 ( p < 0.0001) in laryngeal tumors and improved overall survival of laryngeal cancer patients ( p = 0.045). Chromatin immunoprecipitation, demethylation experiments, and reporter gene assays revealed that the region of methylation loss can function as a CCAAT/enhancer binding protein alpha (CEBPA)-responsive enhancer element regulating TREX2 expression. Conclusions The data highlight a regulatory role of TREX2 DNA methylation for gene expression which might affect incidence and survival of laryngeal cancer. Altered TREX2 protein levels in tumors may affect drug-induced DNA damage repair and provide new tailored therapies. Electronic supplementary material The online version of this article (10.1186/s13148-019-0666-5) contains supplementary material, which is available to authorized users.
Colorectal cancer (CRC) presents as a very heterogeneous disease which cannot sufficiently be characterized with the currently known genetic and epigenetic markers. To identify new markers for CRC we scrutinized the methylation status of 231 DNA repair-related genes by methyl-CpG immunoprecipitation followed by global methylation profiling on a CpG island microarray, as altered expression of these genes could drive genomic and chromosomal instability observed in these tumors. We show for the first time hypermethylation of MMP9, DNMT3A and LIG4 in CRC which was confirmed in two CRC patient groups with different ethnicity. DNA ligase IV (LIG4) showed strong differential promoter methylation (up to 60%) which coincided with downregulation of mRNA in 51% of cases. This functional association of LIG4 methylation and gene expression was supported by LIG4 re-expression in 5-aza-2'-deoxycytidine-treated colon cancer cell lines, and reduced ligase IV amounts and end-joining activity in extracts of tumors with hypermethylation. Methylation of LIG4 was not associated with other genetic and epigenetic markers of CRC in our study. As LIG4 is located on chromosome 13 which is frequently amplified in CRC, two loci were tested for gene amplification in a subset of 47 cases. Comparison of amplification, methylation and expression data revealed that, in 30% of samples, the LIG4 gene was amplified and methylated, but expression was not changed. In conclusion, hypermethylation of the LIG4 promoter is a new mechanism to control ligase IV expression. It may represent a new epigenetic marker for CRC independent of known markers.
The onset of numerous cancers is strongly associated with exposure to genotoxic agents and is counteracted by cellular DNA repair mechanisms. However, the tumorigenic potential of genotoxic carcinogens varies widely among individuals. It is still uncertain which genetic and epigenetic traits shape cancer onset and progression in the general population. While genetic aberrations in DNA repair genes have been linked to cancer risk, less is known about the importance of epigenetics for the regulation of these genes. In order to identify DNA methylation alterations in laryngeal cancer we carried out targeted DNA methylation analysis at single CpG sites via mass spectrometry. We focused our analysis on five DNA repair-associated gene loci previously found to be altered in head and neck squamous cell carcinoma. We report loss of DNA methylation at the three prime repair exonuclease 2 (TREX2) gene locus in laryngeal cancer (n=161) and adjacent normal tissue (n=58) samples of patients from a German population-based case-control study. Following screening of tumor tissues from Chinese colorectal cancer patients as well as previously published data from the Cancer Genome Atlas (TCGA), we identified TREX2 methylation loss as a frequent trait in multiple cancers. We further characterized the regulatory activity of the affected TREX2 site using chromatin immunoprecipitation and luciferase reporter assays in cell models from different tumor types. Differential TREX2 methylation affects a CCAAT/enhancer binding protein alpha (CEBPA) binding site serving as a gene enhancer which drives the expression of TREX2 from a previously uncharacterized gene promoter. We also observed a strong association between TREX2 methylation and TREX2 protein expression determined via immunohistochemistry in laryngeal tumors. Finally, we found a significant association between overall survival and loss of TREX2 methylation in laryngeal cancer, with TREX2 methylation loss being a protective factor. Our findings highlight a profound regulatory role of epigenetic mechanisms for TREX2 in tumors, and underline the usefulness of TREX2 DNA methylation as a biomarker for patient stratification. Citation Format: Christoph Weigel, Jittiporn Chaisaingmongkol, Christine Kuhmann, Irene Santi, Volker Winkler, Olga Bogatyrova, Justo L. Bermejo, Tsun L. Chan, Felix Lasitschka, Manfred H. Bohrer, Alexander Marx, Frank Autschbach, Roland Heyni-von Haußen, Gerhard Dyckhoff, Klaus-Wolfgang Delank, Karl Hoermann, Burkard M. Lippert, Gerald Baier, Andreas Dietz, Christopher C. Oakes, Christoph Plass, Heiko Becher, Peter Schmezer, Heribert Ramroth, Odilia Popanda. DNA methylation loss at an enhancer site of the DNA repair gene TREX2 is an epigenetic feature in multiple cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3364. doi:10.1158/1538-7445.AM2017-3364
Human endonuclease VIII-like1 (NEIL1) encodes a DNA glycosylase which is suggested to be involved in the repair of oxidative damage in association with DNA replication and transcription. We have recently demonstrated that hypermethylation of the NEIL1 gene promoter occurs in more than two third of head and neck squamous cell carcinoma (HNSCC) patients, and that this promoter hypermethylation correlated with a decreased expression both on the mRNA and protein levels. Further experiments were performed to investigate the relevance and functional consequence of these observations. Demethylation experiments using 5-aza-2′-deoxycytidine and DNMT1 knockdown demonstrated an up to 15-fold re-expression of NEIL1 mRNA in cultured HNSCC cells which initially carried hypermethylated promoter regions. The relevance of promoter methylation for NEIL1 expression was further studied by a luciferase promoter assay using CpG-free pCpGL vector constructs in 293T cells. A promoter construct covering the 5′ region of NEIL1 was treated with CpG methyltransferase (M.SssI) or remained untreated. Luciferase expression of the methylated NEIL1 promoter construct was significantly reduced compared to the unmethylated construct (p<0.01, T-Test). Furthermore, NEIL1/2 glycosylase activity was measured in vitro using a bubble structure substrate containing 5-OHU and by quantifying the cleavage products. Nuclear extracts of HNSCC tissue samples which carried NEIL1 promoter hypermethylation showed a more than 2-fold decrease in NEIL1/2 activity when compared to extracts prepared from normal tissue samples without promoter hypermethylation. Taken together, the presented results strengthen the hypothesis that promoter hypermethylation can reduce NEIL1 gene expression and can lead to a significant loss of NEIL1/2 glycosylase activity in HNSCC tissue. NEIL1 hypermethylation may alter the cellular DNA repair capacity and thus play a role in modulating the response to DNA damaging therapies of HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4032. doi:1538-7445.AM2012-4032
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