[Cell Cycle 5:19, 2220-2222, 1 October 2006]; ©2006 Landes Bioscience
Epigenetic Activation of Tumor Suppressor MicroRNAs in Human Cancer Cells Perspective
ABSTRACT
*Correspondence to: Peter A. Jones; 1441 Eastlake Avenue; Los Angeles, California 90089 USA; Tel.: 323.865.0816; Fax: 323.865.0102; Email:
[email protected]. usc.edu
ACKNOWLEDGEMENTS
.
MicroRNAs (miRNAs) are ~22 nucleotide non-coding RNAs that function as endogenous silencers of target genes. Now over 300 miRNAs have been identified in the human genome, and each miRNA is predicted to control hundreds of gene targets. miRNAs are expressed in a tissue specific manner, and play important roles in cell proliferation, apoptosis, and differentiation during animal development.1-4 Moreover, recent studies have shown a distinct connection between aberrant expression of miRNAs and the development of cancer.5,6 So far, most studies in cancer have focused on only protein-coding genes as therapeutic targets. However, the discovery of miRNA genes strongly suggests that many important genes involved in oncogensis can be derived from non-coding regions that consist of approximately 98% of the human genome. In animals, miRNA genes are generally transcribed in the nucleus by RNA polymerase II (pol II) to form long primary transcripts (pri-miRNAs), which are capped with 7-methylguanosine and polyadenylated. The nuclear RNase III enzyme Drosha and its cofactor, Pasha process pri-miRNAs into ~60 nt precursor miRNAs (pre-miRNAs) which can form an imperfect stem-loop structure. Pre-miRNAs are transported into the cytoplasm and subsequently cleaved by the cytoplasmic RNase III enzyme Dicer into mature miRNAs. Mature miRNAs are then loaded into the RNA interference effector complex RISC (RNA-induced silencing complex), where miRNAs can downregulate specific gene products by translational repression via binding to partially complementary sequences in the 3' untranslated regions (3' UTR) of the target mRNAs or by directing mRNA degradation via binding to perfect complementary sequences (Fig. 1).2,7 miRNA sequences and predicted target genes can be analyzed using miRBase (http://microrna.sanger.ac.uk), University of California at Santa Cruz Human Genome Browser (http://genome.cse.ucsc.edu), and Human microRNA Targets (http://www.microrna.org). Although the biological importance of miRNAs is becoming clearer, the regulation of miRNA expression is not fully understood. Many miRNAs are located within the introns of protein-coding genes, and the miRNA expression is therefore coordinately regulated with the expression of the host gene.8 However, some miRNAs are not located within known genes in the same orientation, suggesting that they may have their own promoters. Since miRNAs are expressed in a tissue and tumor specific manner, we expect that some miRNAs are subject to epigenetic control. We have recently shown that some miRNAs can be controlled by epigenetic alterations similar to regular protein-coding genes in human cancer cells, and that activation of tumor suppressor miRNAs by chromatin modifying drugs may be beneficial for anticancer therapy.
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This work was supported by grants from National Institute of Health grant RO1 CA 82422 (P.A.J.) and the Uehara Memorial Foundation of Japan (Y.S.).
INTRODUCTION
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microRNA, DNA methylation, histone modification, epigenetic therapy of cancer
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KEY WORDS
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Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=3340
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Original manuscript submitted: 08/30/06 Manuscript accepted: 08/30/06
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Department of Urology, Biochemistry and Molecular Biology; Norris Comprehensive Cancer Center; University of Southern California; Los Angeles, California USA
MicroRNAs (miRNAs) are small non-coding RNAs that function as endogenous posttranscriptional silencers of target genes. miRNAs are expressed in a tissue specific manner and play important roles in cell proliferation, apoptosis and differentiation during animal development. Recent studies have shown a distinct connection between aberrant expression of miRNAs and the development of cancer, suggesting that miRNAs can be potential therapeutic targets. Our recent study has shown that some miRNAs are controlled by epigenetic alterations such as DNA methylation and histone modification in human cancer cells. Activation of tumor suppressor miRNAs by chromatin modifying drugs may cause downregulation of target oncogenes and could be a novel strategy for the prevention and treatment of human cancer.
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Yoshimasa Saito Peter A. Jones*
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Epigenetic Activation of miRNAs
ROLE OF miRNAS IN CANCER Calin et al.9 have shown that human miRNA genes are frequently located in genomic regions involved in loss of heterozygosity, amplification or breakpoints in cancers, suggesting a link between aberrant expression of miRNAs and the development of cancer. Moreover, Lu et al.6 have revealed that miRNA profiles can be used to classify the developmental lineages and differentiation stages of the tumors. Interestingly, miRNA expression profiles are more accurate for tumor classification than conventional mRNA profiles. They have also shown that most of the miRNAs have lower expression levels in tumors compared to normal tissues, while some miRNAs are upregulated or remain unchanged. Other studies have also shown that some miRNAs are downregulated in various human cancers, indicating that they may function as tumor suppressors.10-12 Indeed, let-7, which is downregulated in lung cancers, targets a critical oncogene RAS,13 and miR-15 and 16, which are downregulated in chronic lymphocytic leukemias, target an antiapoptotic factor BCL2.14 We have demonstrated that miR-127 is highly expressed in normal prostate and bladder tissues, but is remarkably downregulated or silenced in the corresponding tumors. In addition, the protooncogene BCL6 has been identified as one of the targets of miR-127, suggesting that miR-127 has a role as a tumor suppressor.15 Although most of miRNA genes show reduced expression during oncogensis, some miRNA genes are overexpressed in cancers. miR-155 and its host gene BIC are highly expressed in several types of B-cell lymphoma.16 The miR-17-92 cluster, which is located on chromosome 13q31, is activated by the oncogene c-Myc and is highly expressed in B-cell lymphoma and lung cancer.17-19 These studies indicate that some miRNAs may have roles as oncogenes and accelerate the development of cancer. Thus, it is now obvious that miRNAs have critical roles in the underlying mechanism of oncogenesis. Furthermore, recent studies have demonstrated that miRNA expression signatures are associated with prognostic factors and disease progression in chronic lymphocytic leukemia20 and lung cancer,21 indicating that the expression profiling of miRNAs could be a powerful biological marker for diagnosis and prognosis of patients with cancer.
EPIGENETIC REGULATION OF miRNAS IN CANCER CELLS
Figure 1. A model for activation of tumor suppressor miRNAs by chromatin modifying drugs in human cancer cells. Tumor suppressor miRNA genes are silenced by DNA hypermethylation and closed chromatin structure around their promoter regions in human cancer cells. Chromatin modifying drugs such as inhibitors of DNA methylation and HDAC can activate transcription of pri-miRNAs which are capped with 7-methylguanosine (Gppp) and polyadenylated (AAAAA). Drosha and its cofactor, Pasha process pri-miRNAs into pre-miRNAs. Pre-miRNAs are transported into the cytoplasm and subsequently cleaved by Dicer into mature miRNAs. Mature miRNAs are then loaded into the RNA interference effector complex RISC, where miRNAs can downregulate target oncogenes by translational repression. ORF, open reading frame.
Although miRNAs can have large-scale effects through regulation of a variety of genes in human diseases including cancer, the regulatory mechanisms controlling miRNA expression remain to be elucidated. Recent studies have shown that miRNA genes are generally transcribed by RNA pol II and the structure of pri-miRNA has 7-methylguanosine cap and poly(A) tail as same as regular proteincoding genes.7 Moreover, many miRNAs are expressed in a tissue and tumor specific manner. These findings lead us to hypothesize that some Figure 2. Activation of protein-coding and non-coding genes that function as tumor suppressors miRNAs can be regulated by epigenetic alterations by epigenetic therapy. Epigenetic therapy with chromatin modifying drugs such as inhibitors of such as DNA methylation and histone modification DNA methylation and HDAC can activate both protein-coding and non-coding genes including miRNAs that function as tumor suppressors. Activation of tumor suppressor miRNAs may subsesimilar to regular protein-coding genes. quently cause downregulation of target oncogenes. DNA methylation and histone modification play critical roles in chromatin remodeling and general regulation of gene expression in mammalian development of T24 bladder cancer cells with DNA demethylating agent and and in human diseases, such as cancer.22 We have shown that ~5% histone deacetylase (HDAC) inhibitor. In particular, miR-127, which of human miRNAs are upregulated more than 3 fold by treatment is embedded in a CpG island, is remarkably induced by a decrease in www.landesbioscience.com
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DNA methylation levels and an increase in active histone marks around the promoter region of the miR-127 gene.15 These findings suggest that some miRNA genes are controlled by epigenetic alterations in their promoter regions and can be activated by inhibitors of DNA methylation and HDAC. As shown in Figure 1, some tumor suppressor miRNA genes might be silenced by DNA hypermethylation and closed chromatin structure around their promoter regions in human cancer cells. Chromatin modifying drugs such as inhibitors of DNA methylation and HDAC can reduce DNA methylation level and open chromatin structure, resulting in transcriptional activation of these pri-miRNAs. Overexpression of pri-miRNAs would lead to increased level of mature miRNAs by processing with Drosha/Pasha complex and Dicer. These miRNAs can potentially regulate expression of target genes that are important in oncogenesis. Besides miR-127, there are several potential miRNAs that may be under the epigenetic control. Further studies regarding epigenetic regulation of miRNAs are now in progress.
POTENTIAL ANTICANCER THERAPY THROUGH EPIGENETIC REGULATION OF miRNAS
The distinct connection between aberrant expression of miRNAs and the development of cancer suggests that miRNAs can be potential therapeutic targets. A recent study has shown that chemically engineered oligonucleotides, termed ‘antagomirs’ are specific inhibitors of endogenous miRNAs in mice, which could be used to silence oncogenic miRNAs such as the miR-17-92 cluster for anticancer therapy.23 On the other hand, if tumor suppressor miRNAs are downregulated or silenced in cancer cells, activation of these miRNAs may be beneficial for anticancer therapy. Epigenetic therapy with chromatin modifying drugs such as inhibitors of DNA methylation and HDAC has clinical promise as an anticancer therapy. 5-aza-2'-deoxycytidine and 5-azacytidine are DNA methylation inhibitors that have been widely studied and recently approved by the FDA for the treatment of myelodysplastic syndrome (MDS). Many HDAC inhibitors are also under clinical trials.24 In our current study, we have proposed a novel effect of epigenetic therapy with inhibitors of DNA methylation and HDAC to activation of some miRNAs. Activation of tumor suppressor miRNAs such as miR-127 by chromatin modifying drugs may have an anticancer effect through downregulation of their target oncogenes. So far, we have mainly focused on the epigenetic regulation of protein-coding genes that function as tumor suppressors for anticancer therapy, however, our recent study indicates that non-coding genes including miRNAs are also under the epigenetic regulation. This discovery opens up a new field for the therapeutic targets of cancer. Epigenetic therapy with chromatin modifying drugs can activate both protein-coding and non-coding genes including miRNAs that function as tumor suppressors. Activation of tumor suppressor miRNA genes would subsequently cause downregulation of target oncogenes (Fig. 2). Since the number of miRNA genes is increasing and the expression of many miRNAs is reduced in cancer, there could be a large number of potential targets. Further studies are necessary to verify the possibility that epigenetic regulation of miRNAs by chromatin modifying drugs can be a novel strategy for prevention and treatment of human cancer.
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