DNA甲基化与癌症

DNA甲基化是重要的表观遗传修饰,主要发生在DNA的CpG岛. DNA的甲基化通过DNA甲基转移酶（DNA methyltransferases, DNMTs）完成. DNA甲基化参与了细胞分化、基因组稳定性、X染色体失活、基因印记等多种细胞生物学过程.单基因水平及基因组范围内的DNA甲基化改变在肿瘤发生发展中亦发挥重要作用. 抑癌基因的异常甲基化引起的表达抑制,可导致肿瘤细胞的增殖失控和侵袭转移,并参与肿瘤组织的血管生成过程.在许多肿瘤的研究中都发现了基因组整体DNA低甲基化所导致的染色体不稳定性. 本文从DNA的异常高甲基化和低甲基化两方面论述了DNA甲基化在细胞恶变发生发展过程中的改变及其影响,并阐述了DNA甲基化改变在肿瘤诊断和治疗中的作用.     

RFTS-deleted DNMT1 enhances tumorigenicity with focal hypermethylation and global hypomethylation

Site-specific hypermethylation of tumor suppressor genes accompanied by genome-wide hypomethylation are epigenetic hallmarks of malignancy. However, the molecular mechanisms that drive these linked changes in DNA methylation remain obscure. DNA methyltransferase 1 (DNMT1), the principle enzyme responsible for maintaining methylation patterns is commonly dysregulated in tumors. Replication foci targeting sequence (RFTS) is an N-terminal domain of DNMT1 that inhibits DNA-binding and catalytic activity, suggesting that RFTS deletion would result in a gain of DNMT1 function. However, a substantial body of data suggested that RFTS is required for DNMT1 activity. Here, we demonstrate that deletion of RFTS alters DNMT1-dependent DNA methylation during malignant transformation. Compared to full-length DNMT1, ectopic expression of hyperactive DNMT1-ΔRFTS caused greater malignant transformation and enhanced promoter methylation with condensed chromatin structure that silenced DAPK and DUOX1 expression. Simultaneously, deletion of RFTS impaired DNMT1 chromatin association with pericentromeric Satellite 2 (SAT2) repeat sequences and produced DNA demethylation at SAT2 repeats and globally. To our knowledge, RFTS-deleted DNMT1 is the first single factor that can reprogram focal hypermethylation and global hypomethylation in parallel during malignant transformation. Our evidence suggests that the RFTS domain of DNMT1 is a target responsible for epigenetic changes in cancer.     

Cancer genes hypermethylated in human embryonic stem cells

Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs) in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation.     

DNA methylation of mobile genetic elements in human cancers

Mobile genetic elements are responsible for half of the human genome, creating the host genomic instability or variability through several mechanisms. Two types of abnormal DNA methylation in the genome, hypomethylation and hypermethylation, are associated with cancer progression. Genomic hypermethylation has been most often observed on the CpG islands around gene promoter regions in cancer cells. In contrast, hypomethylation has been observed on mobile genetic elements in the cancer cells. It is recently considered that the hypomethylation of mobile genetic elements may play a biological role in cancer cells along with the DNA hypermethylation on CpG islands. Growing evidence has indicated that mobile genetic elements could be associated with the cancer initiation and progression through the hypomethylation. Here we review the recent progress on the relationship between DNA methylation and mobile genetic elements, focusing on the hypomethylation of LINE-1 and HERV elements in various human cancers and suggest that DNA hypomethylation of mobile genetic elements could have potential to be a new cancer therapy target in the future.     

Antiproliferative effects of DNA methyltransferase 3B depletion are not associated with DNA demethylation

Silencing of genes by hypermethylation contributes to cancer progression and has been shown to occur with increased frequency at specific genomic loci. However, the precise mechanisms underlying the establishment and maintenance of aberrant methylation marks are still elusive. The de novo DNA methyltransferase 3B (DNMT3B) has been suggested to play an important role in the generation of cancer-specific methylation patterns. Previous studies have shown that a reduction of DNMT3B protein levels induces antiproliferative effects in cancer cells that were attributed to the demethylation and reactivation of tumor suppressor genes. However, methylation changes have not been analyzed in detail yet. Using RNA interference we reduced DNMT3B protein levels in colon cancer cell lines. Our results confirm that depletion of DNMT3B specifically reduced the proliferation rate of DNMT3B-overexpressing colon cancer cell lines. However, genome-scale DNA methylation profiling failed to reveal methylation changes at putative DNMT3B target genes, even in the complete absence of DNMT3B. These results show that DNMT3B is dispensable for the maintenance of aberrant DNA methylation patterns in human colon cancer cells and they have important implications for the development of targeted DNA methyltransferase inhibitors as epigenetic cancer drugs.     

Regulation of MDA-MB-231 cell proliferation by GSK-3β involves epigenetic modifications under high glucose conditions

Hyperglycemia is a critical risk factor for development and progression of breast cancer. We have recently reported that high glucose induces phosphorylation of histone H3 at Ser 10 as well as de-phosphorylation of GSK-3β at Ser 9 in MDA-MB-231 cells. Here, we elucidate the mechanism underlying hyperglycemia-induced proliferation in MDA-MB-231 breast cancer cells. We provide evidence that hyperglycemia led to increased DNA methylation and DNMT1 expression in MDA-MB-231 cells. High glucose condition led to significant increase in the expression of PCNA, cyclin D1 and decrease in the expression of PTPN 12, p21 and PTEN. It also induced hypermethylation of DNA at the promoter region of PTPN 12, whereas hypomethylation at Vimentin and Snail. Silencing of GSK-3β by siRNA prevented histone H3 phosphorylation and reduced DNMT1 expression. We show that chromatin obtained after immunoprecipitation with phospho-histone H3 was hypermethylated under high glucose condition, which indicates a cross-talk between DNA methylation and histone H3 phosphorylation. ChIP-qPCR analysis revealed up-regulation of DNMT1 and metastatic genes viz. Vimentin, Snail and MMP-7 by phospho-histone H3, which were down-regulated upon GSK-3β silencing. To the best of our knowledge, this is the first report which shows that interplay between GSK-3β activation, histone H3 phosphorylation and DNA methylation directs proliferation of breast cancer cells.     

DNMT cooperativity--the developing links between methylation, chromatin structure and cancer

Controversy has reigned for some time over the biological connection between DNA methylation and cancer. For this reason, the methylation mechanism responsible for increased cancer risk has received greater attention in recent years. Tumor suppressor genes are often hypermethylated resulting in gene silencing. Although some have questioned this interpretation of the link between methylation and cancer, it appears that both hypermethylation and hypomethylation events can create epigenetic changes that can contribute to cancer development. Recent studies have shown that the methyltransferases DNMT1 and DNMT3b cooperatively maintain DNA methylation and gene silencing in human cancer cells. Disruption of the human DNMT3b only slightly reduces the overall global DNA methylation; however, demethylation was markedly potentiated when both DNMT1 and DNMT3b were simultaneously deleted. The results to these experiments provide compelling evidence towards a role for DNA methylation in cancer. This review discusses the current understanding of cancer-epigenetic information and highlights recent studies that connect the methylation machinery and chromatin remodelling with cancer susceptibility.     

Genome-wide hypomethylation in cancer may be a passive consequence of transformation

Epigenetics describes the study of stable, reversible alterations to the genome that affect gene expression and genome function, the most studied mechanisms are DNA methylation and histone modifications. Over recent years there has been rapid progress to elucidate the nature and role of the mechanisms involved in promoter hypermethylation during carcinogenesis, however, the mechanism behind one of the earliest epigenetic observations in cancer, genome-wide hypomethylation, remains unclear. Current evidence is divided between the hypotheses that hypomethylation is either an important early cancer-causing aberration or that it is a passive inconsequential side effect of carcinogenesis. With recent discoveries of gene–body methylation, fast cyclic methylation of hormone dependent genes and candidate proteins involved in DNA demethylation elucidation of the role of hypomethylation and the mechanism behind it appears ever closer. With the burgeoning use of DNA methyltransferase inhibitors as a cancer therapy there is an increased need to understand the mechanisms and importance of genome-wide hypomethylation in cancer. This review will discuss the timing and potential causes of genomic hypomethylation during carcinogenesis and will propose a way forward to understand the underlying mechanisms.     

Stage-specific alterations of DNA methyltransferase expression, DNA hypermethylation, and DNA hypomethylation during prostate cancer progression in the transgenic adenocarcinoma of mouse prostate model

We analyzed DNA methyltransferase (Dnmt) protein expression and DNA methylation patterns during four progressive stages of prostate cancer in the transgenic adenocarcinoma of mouse prostate (TRAMP) model, including prostatic intraepithelial neoplasia, well-differentiated tumors, early poorly differentiated tumors, and late poorly differentiated tumors. Dnmt1, Dnmt3a, and Dnmt3b protein expression were increased in all stages; however, after normalization to cyclin A to account for cell cycle regulation, Dnmt proteins remained overexpressed in prostatic intraepithelial neoplasia and well-differentiated tumors, but not in poorly differentiated tumors. Restriction landmark genomic scanning analysis of locus-specific methylation revealed a high incidence of hypermethylation only in poorly differentiated (early and late) tumors. Several genes identified by restriction landmark genomic scanning showed hypermethylation of downstream regions correlating with mRNA overexpression, including p16INK4a, p19ARF, and Cacna1a. Parallel gene expression and DNA methylation analyses suggests that gene overexpression precedes downstream hypermethylation during prostate tumor progression. In contrast to gene hypermethylation, genomic DNA hypomethylation, including hypomethylation of repetitive elements and loss of genomic 5-methyldeoxycytidine, occurred in both early and late stages of prostate cancer. DNA hypermethylation and DNA hypomethylation did not correlate in TRAMP, and Dnmt protein expression did not correlate with either variable, with the exception of a borderline significant association between Dnmt1 expression and DNA hypermethylation. In summary, our data reveal the relative timing of and relationship between key alterations of the DNA methylation pathway occurring during prostate tumor progression in an in vivo model system.     

HOP/OB1/NECC1 promoter DNA is frequently hypermethylated and involved in tumorigenic ability in esophageal squamous cell carcinoma

Promoter DNA hypermethylation with gene silencing is a common feature of human cancer, and cancer-prone methylation is believed to be a landmark of tumor suppressor genes (TSG). Identification of novel methylated genes would not only aid in the development of tumor markers but also elucidate the biological behavior of human cancers. We identified several epigenetically silenced candidate TSGs by pharmacologic unmasking of esophageal squamous cell carcinoma (ESCC) cell lines by demethylating agents (5-aza-2'-deoxycitidine and trichostatin A) combined with ESCC expression profiles using expression microarray. HOP/OB1/NECC1 was identified as an epigenetically silenced candidate TSG and further examined for (a) expression status, (b) methylation status, and (c) functional involvement in cancer cell lines. (a) The HOP gene encodes two putative promoters (promoters A and B) associated with two open reading frames (HOPalpha and HOPbeta, respectively), and HOPalpha and HOPbeta were both down-regulated in ESCC independently. (b) Promoter B harbors dense CpG islands, in which we found dense methylation in a cancer-prone manner (55% in tumor tissues by TaqMan methylation-specific PCR), whereas promoter A does not harbor CpG islands. HOPbeta silencing was associated with DNA methylation of promoter B in nine ESCC cell lines tested, and reactivated by optimal conditions of demethylating agents, whereas HOPalpha silencing was not reactivated by such treatments. Forced expression of HOP suppressed tumorigenesis in soft agar in four different squamous cell carcinoma cell lines. More convincingly, RNA interference knockdown of HOP in TE2 cells showed drastic restoration of the oncogenic phenotype. In conclusion, HOP is a putative TSG that harbors tumor inhibitory activity, and we for the first time showed that the final shutdown process of HOP expression is linked to promoter DNA hypermethylation under the double control of the discrete promoter regions in cancer.     

Radiation-induced epigenetic alterations after low and high LET irradiations

Epigenetics, including DNA methylation and microRNA (miRNA) expression, could be the missing link in understanding radiation-induced genomic instability (RIGI). This study tests the hypothesis that irradiation induces epigenetic aberrations, which could eventually lead to RIGI, and that the epigenetic aberrations induced by low linear energy transfer (LET) irradiation are different than those induced by high LET irradiations. GM10115 cells were irradiated with low LET X-rays and high LET iron (Fe) ions and evaluated for DNA damage, cell survival and chromosomal instability. The cells were also evaluated for specific locus methylation of nuclear factor-kappa B (NFκB), tumor suppressor in lung cancer 1 (TSLC1) and cadherin 1 (CDH1) gene promoter regions, long interspersed nuclear element 1 (LINE-1) and Alu repeat element methylation, CpG and non-CpG global methylation and miRNA expression levels. Irradiated cells showed increased micronucleus induction and cell killing immediately following exposure, but were chromosomally stable at delayed times post-irradiation. At this same delayed time, alterations in repeat element and global DNA methylation and miRNA expression were observed. Analyses of DNA methylation predominantly showed hypomethylation, however hypermethylation was also observed. We demonstrate that miRNA expression levels can be altered after X-ray irradiation and that these miRNA are involved in chromatin remodeling and DNA methylation. A higher incidence of epigenetic changes was observed after exposure to X-rays than Fe ions even though Fe ions elicited more chromosomal damage and cell killing. This distinction is apparent at miRNA analyses at which only three miRNA involved in two major pathways were altered after high LET irradiations while six miRNA involved in five major pathways were altered after low LET irradiations. This study also shows that the irradiated cells acquire epigenetic changes suggesting that epigenetic aberrations may arise in the cell without initiating chromosomal instability.     

DNA hypermethylation as a chemotherapy target

Epigenetics refers to partially reversible, somatically inheritable, but DNA sequence-independent traits that modulate gene expression, chromatin structure, and cell functions such as cell cycle and apoptosis. DNA methylation is an example of a crucial epigenetic event; aberrant DNA methylation patterns are frequently found in human malignancies. DNA hypermethylation and the associated expression silencing of tumor suppressor genes represent a hallmark of neoplastic cells. The cancer methylome is highly disrupted, making DNA methylation an excellent target for anti-cancer therapies. Several small synthetic and natural molecules, are able to reverse the DNA hypermethylation through inhibition of DNA methyltransferase (DNMT). DNMT is the enzyme catalyzing the transfer of methyl groups to cytosines in genomic DNA. These reagents are studied intensively in cell cultures, animal models, and clinical trials for potential anti-cancer activities. It was found that accompanying DNA demethylation is a dramatic reactivation of the silenced genes and inhibition of cancer cell proliferation, promotion of cell apoptosis, or sensitization of cells to other chemotherapeutic reagents. During the last few decades, an increasing number of DNMT inhibitors (DNMTi) targeting DNA methylation have been developed to increase efficacy with reduced toxicity. This review provides an update on new findings on cancer epigenetic mechanisms, the development of new DNMTi, and their application in the clinical setting. Current challenges, potential solutions, and future directions concerning the development of DNMTi are also discussed in this review.     

前列腺癌的DNA甲基化及其临床应用

目前认为恶性肿瘤的形成是遗传和表观遗传机制共同作用的结果。表观遗传机制包括DNA甲基化、组蛋白修饰和miRNA。DNA异常甲基化(高甲基化和低甲基化)是前列腺癌最具特征的表观遗传改变, 它能够导致基因组不稳定, 调控基因的异常表达, 在前列腺癌的形成和发展中起到重要作用。同时, DNA甲基化作为前列腺癌表观遗传研究的一个热点, 为临床前列腺癌的早期诊断、预后评估及药物治疗提供新的方法和途径。文章根据前列腺癌的DNA高甲基化和低甲基化的最新研究成果阐述了前列腺癌形成的表观遗传学机制, 并且讨论了它们在前列腺癌临床转化方面的最新研究进展。