Bodo Brueckner

The Human let-7a-3 Locus Contains an Epigenetically Regulated MicroRNA Gene with Oncogenic Function

MicroRNAs (miRNAs) are small noncoding RNAs that repress their target mRNAs by complementary base pairing and induction of the RNA interference pathway. It has been shown that miRNA expression can be regulated by DNA methylation and it has been suggested that altered miRNA gene methylation might contribute to human tumorigenesis. In this study, we show that the human let-7a-3 gene on chromosome 22q13.31 is associated with a CpG island. Let-7a-3 belongs to the archetypal let-7 miRNA gene family and was found to be methylated by the DNA methyltransferases DNMT1 and DNMT3B. The gene was heavily methylated in normal human tissues but hypomethylated in some lung adenocarcinomas. Let-7a-3 hypomethylation facilitated epigenetic reactivation of the gene and elevated expression of let-7a-3 in a human lung cancer cell line resulted in enhanced tumor phenotypes and oncogenic changes in transcription profiles. Our results thus identify let-7a-3 as an epigenetically regulated miRNA gene with oncogenic function and suggest that aberrant miRNA gene methylation might contribute to the human cancer epigenome.

Epigenetic Reactivation of Tumor Suppressor Genes by a Novel Small-Molecule Inhibitor of Human DNA Methyltransferases

DNA methylation regulates gene expression in normal and malignant cells. The possibility to reactivate epigenetically silenced genes has generated considerable interest in the development of DNA methyltransferase inhibitors. Here, we provide a detailed characterization of RG108, a novel small molecule that effectively blocked DNA methyltransferases in vitro and did not cause covalent enzyme trapping in human cell lines. Incubation of cells with low micromolar concentrations of the compound resulted in significant demethylation of genomic DNA without any detectable toxicity. Intriguingly, RG108 caused demethylation and reactivation of tumor suppressor genes, but it did not affect the methylation of centromeric satellite sequences. These results establish RG108 as a DNA methyltransferase inhibitor with fundamentally novel characteristics that will be particularly useful for the experimental modulation of epigenetic gene regulation.

Methylation of human MicroRNA genes in normal and neoplastic cells

MicroRNAs (miRNAs) are small RNA molecules that control gene expression by inhibition of protein translation or by degradation of cognate target mRNAs. Even though strict developmental and tissue-specific regulation appears to be critical for miRNA function, very little is known about the mechanisms governing miRNA gene expression. Several recent studies have shown that miRNA genes can be regulated by DNA methylation and other epigenetic mechanisms. The observation of altered miRNA gene methylation patterns in human cancers also suggested that miRNA gene methylation is functionally relevant for tumorigenesis. We have now performed a comprehensive analysis of miRNA genes and found that about half of these genes are associated with CpG islands and thus represent candidate targets of the DNA methylation machinery. An expanded analysis of several miRNA-associated CpG islands in five cell lines indicated that miRNA gene methylation is detectable at high frequencies, both in normal and malignant cells. Possible explanations for this phenomenon include the specific structure of miRNA genes and/or their requirement for strict expression regulation.

Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines

The DNA methyltransferase inhibitors azacytidine and decitabine represent archetypal drugs for epigenetic cancer therapy. To characterize the demethylating activity of azacytidine and decitabine we treated colon cancer and leukemic cells with both drugs and used array-based DNA methylation analysis of more than 14,000 gene promoters. Additionally, drug-induced demethylation was compared to methylation patterns of isogenic colon cancer cells lacking both DNA methyltransferase 1 (DNMT1) and DNMT3B. We show that drug-induced demethylation patterns are highly specific, non-random and reproducible, indicating targeted remethylation of specific loci after replication. Correspondingly, we found that CG dinucleotides within CG islands became preferentially remethylated, indicating a role for DNA sequence context. We also identified a subset of genes that were never demethylated by drug treatment, either in colon cancer or in leukemic cell lines. These demethylation-resistant genes were enriched for Polycomb Repressive Complex 2 components in embryonic stem cells and for transcription factor binding motifs not present in demethylated genes. Our results provide detailed insights into the DNA methylation patterns induced by azacytidine and decitabine and suggest the involvement of complex regulatory mechanisms in drug-induced DNA demethylation.

DNA methyltransferase inhibitors for cancer therapy.

Aberrant DNA methylation patterns, including hypermethylation of tumor suppressor genes, have been described in many human cancers. These epigenetic mutations can be reversed by DNA methyltransferase inhibitors, which provide novel opportunities for cancer therapy. Clinical concepts for epigenetic therapies are currently being developed by using azanucleosides for the treatment of leukemias and other tumors. These trials will greatly benefit from the inclusion of molecular markers for monitoring epigenetic changes in patients and for maximizing biologic responses. In addition, novel inhibitors need to be developed that result in a direct and specific inhibition of DNA methyltransferase activity. Several recent developments indicate that rational design of small molecule DNA methyltransferase inhibitors is feasible and that this approach can result in the establishment of novel drug candidates. The use of novel DNA methyltransferase inhibitors in clinical trials that allow monitoring of drug-induced DNA methylation changes should provide the foundation for improved epigenetic cancer therapies.

Reactivation of epigenetically silenced genes by DNA methyltransferase inhibitors: basic concepts and clinical applications.

Hypermethylation of tumor suppressor genes is one of the most consistent hallmarks of human cancers. This epigenetic alteration has been associated with gene silencing and thus represents an important pathway for generating loss-of-function mutations. In this review, we survey the available literature on systematic, genome-wide approaches aimed at the identification of epigenetically silenced loci. These studies uncovered a variety of diverse genes, but a common signature for epigenetic reactivation has not been identified. Nevertheless, DNA methyltransferase inhibitors have shown significant clinical benefits, mostly in the therapy of leukemias. Recent analyses revealed substantial drug-induced methylation changes that can now be used as endpoints for the further refinement of clinical treatment schedules. Further optimization of epigenetic cancer therapies should be feasible through the use of novel DNA methyltransferase inhibitors with improved specificity. Rational design of epigenetic inhibitors might provide the foundation for a broader use of these drugs in the treatment of cancer.

Delivery of 5-Azacytidine to Human Cancer Cells by Elaidic Acid Esterification Increases Therapeutic Drug Efficacy

Azacytidine is an established nucleoside drug that is well known for its ability to modulate epigenetic gene regulation by inhibition of DNA methylation. Despite recent advances in the clinical development of azacytidine, the use of the drug is limited by its low bioavailability and dependency on variably expressed nucleoside transporters for cellular uptake. We show here that CP-4200, an elaidic acid derivative of azacytidine, has strong epigenetic modulatory potency in human cancer cell lines, as evidenced by efficient depletion of DNA methyltransferase protein, genome-wide DNA demethylation, and robust reactivation of epigenetically silenced tumor suppressor genes. Importantly, however, the cellular uptake of CP-4200 was substantially less dependent on the nucleoside transporters that are known to be involved in azacytidine uptake. In agreement with this notion, CP-4200 showed a significantly higher antitumoral activity than azacytidine in an orthotopic mouse tumor model for acute lymphocytic leukemia. Together, these data represent a detailed characterization of the CP-4200 mode of action and suggest that elaidic acid modification improves the therapeutic efficacy of azacytidine. Mol Cancer Ther; 9(5); 1256–64. ©2010 AACR.

Epigenetic regulation of depot-specific gene expression in adipose tissue.

In humans, adipose tissue is distributed in subcutaneous abdominal and subcutaneous gluteal depots that comprise a variety of functional differences. Whereas energy storage in gluteal adipose tissue has been shown to mediate a protective effect, an increase of abdominal adipose tissue is associated with metabolic disorders. However, the molecular basis of depot-specific characteristics is not completely understood yet. Using array-based analyses of transcription profiles, we identified a specific set of genes that was differentially expressed between subcutaneous abdominal and gluteal adipose tissue. To investigate the role of epigenetic regulation in depot-specific gene expression, we additionally analyzed genome-wide DNA methylation patterns in abdominal and gluteal depots. By combining both data sets, we identified a highly significant set of depot-specifically expressed genes that appear to be epigenetically regulated. Interestingly, the majority of these genes form part of the homeobox gene family. Moreover, genes involved in fatty acid metabolism were also differentially expressed. Therefore we suppose that changes in gene expression profiles might account for depot-specific differences in lipid composition. Indeed, triglycerides and fatty acids of abdominal adipose tissue were more saturated compared to triglycerides and fatty acids in gluteal adipose tissue. Taken together, our results uncover clear differences between abdominal and gluteal adipose tissue on the gene expression and DNA methylation level as well as in fatty acid composition. Therefore, a detailed molecular characterization of adipose tissue depots will be essential to develop new treatment strategies for metabolic syndrome associated complications.

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.

Abstract 1036: Antiproliferative effects of DNA methyltransferase 3B depletion are not associated with DNA demethylation

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Silencing of genes by DNA 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 after depletion of DNMT3B protein have not been analyzed in detail yet. We performed short- and long-term RNAi knockdown experiments to reduce DNMT3B protein levels in colon cancer cell lines and analyzed genome-wide DNA methylation changes on HumanMethylation27 and HumanMethylation450 Illumina bead chips. Our results confirm that depletion of DNMT3B specifically reduced the proliferation rate of DNMT3B-overexpressing colon cancer cell lines. All colon cancer cell lines tested contain a mutant K-Ras and we conclude that K-Ras status - in contrast to DNMT3B protein overexpression levels - does not impact on anti-proliferative responses after DNMT3B knockdown. However, in contrast to the dramatic alterations of DNA methylation observed in DNMT1; DNMT3B double knockout cells (DKO), 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. 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 1036. doi:1538-7445.AM2012-1036