Epigenetic changes in osteosarcoma

Electronic journal of oncology

Volume 98 • N◦ 7 • juillet 2011 John Libbey Eurotext

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Epigenetic changes in osteosarcoma Article received on September 12, 2010, accepted on December 14, 2010 Reprints: J. Cui

Juncheng Cui1 , Wanchun Wang1 , Zhihong Li1 , Zhaogui Zhang1 , Bei Wu2 , Li Zeng3 1

Central South University, The Second Xiangya Hospital, Department of Orthopedics, Changsha, Hunan, China 2 Central South University, Xiangya Hospital, Department of Orthopedics, Changsha, Hunan, China 3 The First Affiliate Hospital of Nanhua University, Department of Medical Cosmetology, Hengyang, Hunan, China

To cite this article: Cui J, Wang W, Li Z, Zhang Z, Wu B, Zeng L. Epigenetic changes in osteosarcoma. Bull Cancer 2011 ; 98 : E62-E68. doi : 10.1684/bdc.2011.1400.

Abstract. Osteosarcoma is one of the most prevalent primary bone tumors. The pathogenesis and molecular development of this tumor remains elusive. The prognosis is unfavorable due to lack of effective treatment methods. Recent advances in the epigenetics have brought a profound impact on the understanding of molecular mechanisms that lead to osteosarcoma. In this review, we summarized the current literature on

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steosarcoma is one of the most prevalent primary bone tumors. The age distribution of this tumor is bimodal, with peaks in adolescence and above the age of 50 years [1]. The pathogenesis and molecular mechanisms of this tumor remain elusive. Although several effective treatment options are available, local recurrence (4-8%) and postoperative pulmonary metastasis of osteosarcoma (about 30%) are frequent, the prognosis of osteosarcoma is unfavorable [2]. Recent progresses in the epigenetics research have shown that epigenetic changes appear to contribute to the malignant transformation and progression of osteosarcoma, providing a better understanding of the molecular mechanisms of this tumor.

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Key words: osteosarcoma, epigenetics, DNA methylation, histone modifications

DNA methylation DNA methylation refers to the addition of a methyl group to the 5 -carbon of cytosine in CpG sequences, catalyzed by DNA methyltransferases (DNMTs) [4, 5]. A study of the human genome revealed that the distribution of CpG dinucleotides is not random. Some of CpG dinucleotides cluster together to form CpG-rich DNA regions called CpG islands, where methylcytosine residues are often found in CpG islands are 0.5 to 2 kb long and found in the 5 region of approximately 60% of genes [6]. Most CpG islands in actively expressed genes are unmethylated, with the exception of certain imprinted genes and genes on the inactive X chromosomes of females [7, 8]. However, during cancer development, DNA methylation in cancer cells exhibits inverse profiles compared with normal cells [9, 10]. DNA methylation aberrations can occur as either hypermethylation or hypomethylation. Both forms can lead to chromosomal instability and transcriptional gene silencing, both forms have been implicated in a variety of human malignancies, including osteosarcoma.

DNA hypermethylation DNA hypermethylation is a well-investigated epigenetic abnormality seen in several malignancies. Aberrant methylation of gene promoters has been Bull Cancer vol. 98 • N◦ 7 • juillet 2011

doi : 10.1684/bdc.2011.1400

Epigenetics refers to the research of heritable changes in gene expression without the change in gene sequence [3]. It is one of the most rapidly expanding fields in cancer related research. DNA methylation and histone modifications are two important, well-studied epigenetic mechanisms in osteosarcoma. These two processes can act either independently or dependently affecting the gene expression and in turn the tumorigenesis. Here, we provide an overview of the current understanding of epigenetic changes associated with osteosarcoma, and the potential diagnostic and therapeutic applications in osteosarcoma.

epigenetic changes that are thought to contribute to the carcinogenesis of osteosarcoma, and discussed the potential diagnostic and therapeutic applications as well as future areas of research. 

Epigenetic changes in osteosarcoma

hypothesized as an early event in the neoplastic progression [11]. DNA hypermethylation contributes to gene silencing by preventing the binding of activating transcription factors and by attracting repressor complexes that induce the formation of inactive chromatin structures. Numerous tumor suppressor genes have been identified as being regulated by DNA hypermethylation in different types of cancer, such as breast tumor and malignant melanoma [12, 13]. With regard to osteosarcoma, the exact function of most of these aberrantly silenced genes is still unknown; however, a few studies have indicated that some aberrantly silenced genes are closely associated with osteosarcoma progression. Besides, these genes involved in a number of cellular pathways such as cell cycle regulation, apoptosis, signal transduction, tumor cell proliferation and differentiation and growth arrest. For many of these genes, CpG island DNA hypermethylation seems to play a central role in osteosarcoma progression and metastasis, as summarized in table 1.

by demethylases including 5-methylcytosine glycosylase and MBD2b [39], it can causes breakdown of such a defense mechanism, and lead to structural and functional alterations of the genome. There are two types of hypomethylation: global or localized hypomethylation. Global hypomethylation refers to overall decrease in 5-methylcytosine content in the genome. Localized or gene specific hypomethylation denotes to a decrease in cytosine methylation relative to the “normal” methylation level. It can affect several specific regions of the genome, such as the promoter regions of oncogenes or normally highly methylated sequences such as repetitive sequences and oncogenes [40]. Both global hypomethylation and localized hypomethylation have been implicated in human cancer. Genes, which have been identified to be aberrantly hypomethylated in osteosarcoma are summarized in table 2.

Histone modification DNA hypomethylation DNA hypomethylation is another type of methylation change related to epigenetic aberration in many malignancies including osteosarcomas. DNA methylation of normal genomes is a defensive mechanism by which repetitive DNA is transcriptionally silenced to prevent it from propagating [38]. Hypomethylation is catalyzed

The basic structural unit of chromatin is the nucleosome, a complex structure composed of an octamer of four core histones, H2A, H2B, H3 and H4. Histones have been critically implicated in regulation of chromatin structure and gene expression. They comprise a globular domain and a more flexible and charged NH2 terminal, which is called as histone

Table 1. Hypermethylated genes in osteosarcomas. Genes

Common names

Genes ID

Functions

CDK4

Cyclin-dependent kinase 4

1019

Amplification and cell cycle apoptosis [14]

ChM-I

Chondromodulin-I

162840

Growth arrest

[15,16]

EGFR

Epidermal growth factor receptor

1956

Amplifications

[17–19]

GADD45A Growth arrest and DNA-damage-inducible 45 alpha 13197

Apoptosis

[20–22]

IGF2

Insulin-like growth factor 2

3481

Signal transduction

[23,24]

INK4A

Cyclin-dependent kinase inhibitor 2A

1029

Cell cycle regulation

[25–28]

P53

p53 tumor suppressor homologue

100384887 Cell cycle regulation and apoptosis

[29,30]

RASSF1A

RAS association domain family protein 1A

11186

Signal transduction

[31,32]

RECQL4

RecQ protein-like 4

9401

Mutations

[33,34]

RUNX2

Runt-related transcription factor 2

367218

Signal transduction

[35,36]

WIF-1

WNT inhibitory factor 1

11197

Proliferation and differentiation

[37]

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References

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J. Cui, et al. Table 2. Hypomethylated genes in osteosarcomas. Genes

Common names

Genes ID

Functions

References

FOS

FBJ osteosarcoma oncogene

314322

Amplifications

[41,42]

H19

H19, imprinted maternally expressed transcript

283120

Growth arrest

[43]

MYC

V-myc myelocytomatosis viral oncogene homologue

4609

Amplifications

[44–46]

PHLDA2

Pleckstrin homology-like domain, family A, member 2

22113

Apoptosis

[43]

“tail”. These tails are subject to a variety of covalent modifications including acetylation, methylation, phosphorylation, ubiquitylation and sumoylation [47, 48]. These modifications are involved in regulation of gene expression. The most widely studied modifications are histone acetylation and deacetylations. They are regulated by histone acetyltransferases (HAT) and histone deacetylases (HDAC), respectively. It has been recognized in recent years that the HDACs play a fundamental role in keeping the balance between the acetylated and deacetylated state of chromatin [49]. Consequently, considerable effort has been devoted to developing HDAC inhibitors (HDACi), which have been found to induce numerous anti-proliferative effects including apoptosis, cytostasis and differentiation [50]. In osteosarcomas, several genes of biological significance may be potentially regulated by histone modification. One such gene is p21WAF1, which arrests cell growth by inhibiting cyclin/Cdk complexes and can act as tumor suppressor gene by negatively regulating the cell cycle [51]. Decreased expression of the p21WAF1 has been detected in osteosarcoma and is associated with the malignancy [52]. Maeda et al. had shown that p21WAF1 was upregulated by HDACi including TSA and sodium butyrate in MG63 cells. Moreover, the cell cycle analysis revealed that the p21WAF1 arrested MG63 cells in the G2/M phase [53]. Cellular FLICE-inhibitory protein (cellular FLIP) is another gene regulated by histone modification in osteosarcomas. It is an inhibitor of Fas-mediated activation of caspase-8. HDACi including FR901228 were shown to induce downregulation of cellular FLIP through inhibiting generation of FLIP mRNA, The downregulation of cellular FLIP renders Fas-resistant osteosarcoma cell lines become more sensitive to Fasmediated apoptosis [54].

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Interactions between DNA methylation and histone modifications Both DNA methylation and histone acetylation has been suggested to modulate gene expression [55-57]. It has been shown that methylated DNA recruits HDAC through methyl-DNA binding proteins (MBPs). This interaction then recruits HDAC activity to methylated promoters which results in gene silencing [58]. Furthermore, DNMTs can directly recruit HDAC activity to silence gene expression [59, 60]. Cheng et al. have demonstrated that monomethyl histone H3 lysine 27 is expressed in staurosporine-induced apoptotic osteosarcoma cell lines in vitro [61]. Chondromodulin-I (ChM-I) is a vascular endothelial cell growth inhibitor purified from bovine epiphyseal cartilage [62], the expression of this gene is regulated by the binding of Sp3 to the core promoter region, which is inhibited by the methylation of CpG in the target genome in the osteogenic lineage, osteosarcoma cells [15]. The histone tails associated with the hypermethylated promoter region of the ChM-I gene were deacetylated by HDAC2 in three ChM-I- negative osteosarcoma cell lines. Treatment with an HDACi induces the binding of Sp3 in one cell line, which became ChM-I-positive. This process was associated with acetylation instead of the dimethylation of histone H3 at lysine 9 (H3-K9) and the demethylation of the core promoter region. The demethylation was transient. It is gradually replaced by methylation after a rapid recovery of histone deacetylaion [63]. These studies provide evidence that the plasticity of differentiation being regulated by the cell- specific plasticity of epigenetic regulation and provide a rationale for a combined treatment with DNA methylation and HDACi in reversing the epigenetic silencing of key tumor suppressor genes. Bull Cancer vol. 98 • N◦ 7 • juillet 2011

Epigenetic changes in osteosarcoma

Epigenetic changes as biomarkers Early detection is the most efficient way to decrease mortality of osteosarcoma. Specific epigenetic changes have been found occurring early in multistep carcinogenesis of osteosarcomas. These changes have been considered as potential and promising biomarkers for early detection of this tumor. To be clinically applicable, the biomarkers must be readily detectable in clinical specimens such as cancer tissues, serum and body fluids. MTA is a component of histone deacetylation (NuRD) complex, which is associated with ATP-dependent chromatin remodeling and HDAC activity [64]. Park et al. demonstrated in their study that the ezrin and MTA positivity can be additional prognostic markers for osteosarcoma of the jaw [65]. p16INK4a is the G1 cell-cycle checkpoint protein that is responsible for maintaining the balance of cellular proliferation. It is a subject of frequent involvement in osteosarcomas. Importantly, hypermethylation of the p16INK4A promoter is significantly associated with malignancy and metastasis [66]. Maitra et al. examined a series of 38 pediatric osteosarcomas with 16% loss of p16INK4a expression, while absence of p16INK4a expression significantly correlated with decreased survival. These findings suggested that immuno-histochemical analysis of p16INK4a expression in osteosarcomas may be a useful biomarker of prognosis [67]. Furthermore, studies are needed so that new biomarkers may be able not only to prognosticate osteosarcoma patients but also to serve as therapeutic targets and thereby improve survival rates in the routine clinical practice.

Therapeutic targets Unlike genetic events that occur at tumor suppressor genes, epigenetic modifications are heritable and potentially reversible. Therefore, epigenetic modifications can be used as potential therapeutic targets. At present, the most widely investigated drugs can be classified in two types: (1) DNMT inhibitors, such as 5-azacytidine (azacitidine) and 5-aza-2 -deoxycytidine (5-aza-dC), (2) HDACi.

deoxynucleotide triphosphates. Eventually, deoxynucleotide triphosphates can incorporate into RNA (5AC) and/or DNA (5AC and DAC) in place of cytosine [68]. Nucleoside analogue inhibitors can also induce hypomethylation by depletion of cellular DNMTs [69]. Azacitidine is the first DNA hypomethylating agent approved by FDA for the treatment of myelodysplastic syndromes. Marcucci et al. [70] demonstrated a single dose of azacitidine at 75 mg/m2 that is well tolerated in adult population. Myelosuppression is a known side effect of this drug, which may limit their clinical utility. Zebularine is the first orally bioavailable drug in this group, it is chemically stable and significantly less cytotoxic [71]. Other categories drugs are being studied, such as non-nucleoside analogue DNA methylation inhibitors, including epigallocatechin-3-gallate (EGCG), procaine, procainamide and hydralazine, DNMT1 antisense oligonucleotides (MG98).

HDAC inhibitors A number of compounds exhibit HDAC inhibitory activity, which are commonly used including the valproic acid (VPA), the trichostatin A (TSA), the benzamides SNDX-275 (formerly MS-275) and MGCD0103 and the cyclic peptide FK-228. A recent research by Wittenburg et al. demonstrated that pretreatment of OS cell lines with VPA followed by doxorubicin (DOX) resulted in significant growth inhibition and potentiation of apoptosis, associated with a dose-dependent increase in nuclear DOX accumulation [72]. Suberoyl anilide hydroxamic acid (SAHA), which is structurally similar to TSA, is the first approved HDACi for clinical use in cancer patients by the FDA [73]. Kisseberth et al. provided evidence that SAHA induced apoptosis in cells from osteosarcoma and histiocytic sarcoma cell lines in vitro [74]. Blattmann et al. demonstrated that SAHA induced an inhibition of cell proliferation and clonogenic survival and led to a significant radiosensitization in OS cell lines [75]. Nevertheless, as a class there are of limited clinical application due to side effects, such as myelosuppression, fatigue, somnolence and gastrointestinal symptoms [76].

Combination therapy DNMT inhibitors Azacitidine and 5-aza-dC are nucleoside analogue inhibitors known to inhibit DNA methylation. The mechanism of action is that they can be converted to the Bull Cancer vol. 98 • N◦ 7 • juillet 2011

The single epigenetic drug activity currently is limited and associated with significant side effects. Thus, it prompted investigation of the pharmacodynamic interactions between DNMTs and HDACi. The

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combinatorial use of these two agents in the clinic is based on their synergistic effects in reestablishing the expression of tumor suppressor genes. Some hypermethylated genes cannot be transcriptionally reactivated with HDACi (TSA) alone in tumor cells, but sequential treatment with 5-azacytosine nucleosides and subsequently with an HDACi (TSA), results in more potent re-expression of methylated tumor suppressor genes than either agent alone [77]. These results indicate that the combined strategies of anticancer therapy may make these drugs more versatile and result in a synergistic effect on various tumor cells. Additionally, the combination therapy may lead to reduction in side effects and the dose of each individual agent.

Summary and future directions Currently, epigenetic changes in tumors are being extensively studied and genome wide analysis of mutated genes in breast and colorectal cancers has been well-annotated [78]. The advance in the human cancer epigenetic field has encouraged us to undertake osteosarcoma epigenome projects to map its epigenetic modifications. The outcome of these projects will enable us to understand the definitely progressive changes in epigenomic patterns during the development of osteosarcoma. And it is essential for us to master the development of epigenetic markers and therapeutics, which will be very important for the application of these advances to diagnosis, prognosis and therapy of osteosarcoma patients. Furthermore, although the current development of epigenetic diagnosis and therapeutics has been demonstrated to be a promising prospect, the more detailed comprehension of the molecular mechanisms underlying the correlation between reversion of epigenetic changes, gene re-expression, therapeutic response and epigenetic drugs is essential.  Conflict of interest : none.

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